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WO2007041689A2 - Methods of site-specific labeling of molecules and molecules produced thereby - Google Patents

Methods of site-specific labeling of molecules and molecules produced thereby Download PDF

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Publication number
WO2007041689A2
WO2007041689A2 PCT/US2006/039061 US2006039061W WO2007041689A2 WO 2007041689 A2 WO2007041689 A2 WO 2007041689A2 US 2006039061 W US2006039061 W US 2006039061W WO 2007041689 A2 WO2007041689 A2 WO 2007041689A2
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molecule
seq
alpha
agent
protein
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PCT/US2006/039061
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French (fr)
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WO2007041689A3 (en
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Christopher T. Walsh
Jun Yin
Paul D. Straight
Roberto Kolter
Zhe Zhou
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President And Fellows Of Harvard College
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif

Definitions

  • O 6 -alkylguanine-DNA alkyltransferase can be fused to a target protein of interest, followed by the addition of a fluorescently-labeled O 6 -benzylguanine suicide substrate for the AGT.
  • AGT tag is 207 amino acids long and introduces a large amount of steric bulk. While, smaller peptide tags are generally more desirable, they typically lack specificity.
  • cysteine labeling is not at all specific inside cells, and tetracysteine labeling (Griffin, B A et al, Science 281, 269-272, 1998), while better, is still insufficiently specific for most applications and allows only a small set of probes to be introduced.
  • Transglutaminase has also been used to label glutamine side chains in proteins with fluorophores in vitro (Sato, H. et ah, Biochemistry 35, 13072-13080, 1996), however it is relatively promiscuous for peptide and protein substrates, precluding its use in mammalian cells.
  • intein-based methods were described for attaching a wide array of small molecules to proteins including, for example, fluorophores, carbohydrates, oligonucleotides, affinity tags and metal chelators.
  • a major drawback of the intein-based methods is the size of the intern domain itself, which is about 454 amino acids long.
  • Other disadvantages of the intein-based methods include, for example, the length of the reaction which typically requires an overnight incubation.
  • the present invention relates, at least in part, to the discovery of novel peptide substrates for phosphopantetheinyl transferases, such as, for example, Sfp and Acps, which can be used for site-specific labeling of both proteinaceous and non-proteinaceous molecules.
  • the present invention provides methods for site- specific labeling of polypeptides, and labeled polypeptides produced by such methods.
  • the present invention relates to a method of generating a molecule of formula (I):
  • L is a linking group
  • Y is an alpha-helical oligopeptide moiety
  • Z is a second agent; comprising: a) providing a compound of formula (II):
  • Y is the alpha-helical oligopeptide moiety
  • Z is the second agent
  • L is the linking group; and N is a leaving group; under suitable conditions to allow the enzyme or the fragment thereof to attach M q -L to the alpha-helical oligopeptide moiety (Y), thereby generating the compound of formula (I).
  • the present invention provides a method of generating a molecule of formula (VIII):
  • G is a transferred moiety
  • Y is an alpha-helical oligopeptide moiety
  • Z is a second agent, comprising:
  • Y is the alpha-helical oligopeptide moiety
  • Z is the second agent
  • G is the transferred moiety; and N is a leaving group; under suitable conditions to allow the enzyme or the fragment thereof to attach the transferred moiety (G) in formula (X) to the alpha-helical oligopeptide moiety (Y) in formula (IX), thereby to generate the molecule of formula (VIII).
  • the transferred moiety generally includes a first agent linked to a linking group.
  • Alpha-helical oligopeptide moieties that can be used in the methods described herein include those set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:40, and SEQ ID NO:41, and variants thereof, which are capable of being phosphopantetheinylated by an enzyme having phosphopantetheinyl transferase activity, e.g., Sfp and Acps.
  • the first agent used in the methods described herein is a chosen from the group consisting of small molecules moieties (e.g., drug moieties, toxins), protons, haptens, affinity probes, spectroscopic probes, radioactive probes, peptides, non-naturally occurring amino acids, nucleic acids, lipid molecules, radical generating molecules, singlet oxygen generating molecules, polymers, sugars (e.g., monosaccharides, disaccharides, polysaccharides, and other carbohydrate containing moieties), antibodies and antibody fragments, enzymes, enzyme substrates, chelating agents and receptor binding molecules.
  • small molecules moieties e.g., drug moieties, toxins
  • protons e.g., protons, haptens, affinity probes, spectroscopic probes, radioactive probes, peptides, non-naturally occurring amino acids, nucleic acids, lipid molecules, radical generating molecules, singlet oxygen generating molecules, polymers, sugars (e.
  • the second agent used in the methods described herein can either be proteinaceous or non-proteinaceous.
  • proteinaceous second agents include, for example, polypeptides, enzymes, fusion proteins, antibodies and hormones.
  • non-proteinaceous second agents include, but are not limited to, a nucleic acid molecule and derivatives thereof, a carbohydrate and derivatives thereof, and a lipid molecule and derivatives thereof.
  • a second agent may also be a small molecule moiety.
  • the present invention provides a method of generating a protein-small molecule conjugate of formula (XI):
  • L is a linking group
  • Y is an alpha-helical oligopeptide moiety
  • K is a protein; comprising: a) providing a compound of formula (XII):
  • Y is the alpha-helical oligopeptide moiety; and K is the protein; and b) contacting the compound of formula (XII) with an enzyme having phosphopantetheinyl transferase activity or a fragment thereof having phosphopantetheinyl transferase activity, in the presence of a compound of formula (XIII):
  • I is the small molecule; q is an integer greater than 0; L is the linking group; and N is a leaving group; under suitable conditions to allow the enzyme or the fragment thereof to attach the small molecule (Iq) to the alpha-helical oligopeptide moiety (Y) via the linking group (L) thereby generating the protein-small molecule conjugate of formula (XI).
  • XI protein-small molecule conjugate of formula
  • L is a linking group
  • Y is an alpha-helical oligopeptide moiety
  • compositions containing molecules produced by the methods described herein may further include a pharmaceutically acceptable carrier.
  • a method of delivering a second agent such as, for example, a protein, to a desired location in a subject includes for example, administering to a subject a molecule of formula (T), where the first agent is capable of delivering the second agent to the desired location within the subject, where the molecule of formula (T) is:
  • M is a first agent; q is an integer greater than 0; L is a linking group;
  • Y is an alpha-helical oligopeptide moiety
  • Z is a second agent.
  • a method of labeling two target proteins includes (a) providing a first protein and a second protein, where the first protein is linked to at least one alpha helical oligopeptide moiety including an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40, and the second protein is linked to at least one alpha helical oligopeptide moiety including an amino acid sequence set forth in SEQ ID NO:41; and (b) contacting the first protein and the second protein with one or more compounds in the presence of one or more phosphopantetheinyl transferase enzymes chosen from Sfp, Acps, or a fragment thereof having phosphopantetheinyl transferase activity, under conditions such that to allow the enzyme to transfer the one or more compounds to the one or more alpha-helical oligopeptide moieties in step (a).
  • the Sfp enzyme or a fragment thereof having phosphopantetheinyl transferase activity transfers a compound to an alpha-helical oligopeptide moiety having an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40, thereby to label the first protein and the Acps enzyme or a fragment thereof having phosphopantetheinyl transferase activity transfers a compound to an alpha-helical oligopeptide moiety having an amino acid sequence set forth in SEQ ID NO:41, thereby to label the second protein.
  • the compound includes the formula I q -L-N (XIII), where:
  • I is the small molecule; q is an integer greater than 0; L is the linking group; and
  • N is a leaving group
  • the first and the second protein may either be labeled simultaneously or sequentially. Also, the first and the second protein may either be labeled with the same compound or each of the first and second protein may be labeled with different compound.
  • the first and the second protein may either be present in a cell lysate or be expressed on the surface of live cells.
  • Figure 1 depicts Sfp catalyzed PCP or ybbR tag modification at a specific Serine residue by various small molecule-CoA conjugates.
  • Rl depicts biotin-CoA conjugate;
  • R2 depicts fluorescein-CoA conjugate;
  • R3 depicts tertramethylrhodamine- CoA conjugate and
  • R4 depicts Texas Red-CoA conjugate.
  • Figure 2 depicts amino acid sequence alignment of B. subtilis ybbR open reading frame (amino acids 1-299) with the truncated ybbR clones JY503, JY529, JY530 and JY565, selected by phage display. Sequences that match are highlighted. Truncated ybbR amino acid sequences are preceded by a leader peptide followed by the sequence of the phage capsid protein pUI. The Ppant modified Serine residue at position 274 is boxed.
  • Figure 3 depicts results of a Western blot analysis of JY529-His labeled with biotin by Sfp catalyzed biotin-CoA modification.
  • a Ser274Ala mutant of JY529-His was used as a control and the Western blot was probed with streptavidin-HRP.
  • Figure 4 depicts localization of the Ppant modified serine in ybbR JY529-His using Q-FTMS and tandem mass spectrometry.
  • Figure 4a is the charge state distribution for intact Ppant modified JY529-His (8,853.37 Da).
  • Figure 4b depicts mass selection of the 9+ charge state of Ppant modified JY529-His.
  • Figure 4c shows fragment ion profiles for JY529-His.
  • Figure 4d depicts the y23 ion, generated using IRMPD. This ion is 80 Da greater in mass, consistent with phosphate retention.
  • Figure 4e shows the y20 ion generated using IRMPD. Similar to the previous ion, this species is 80 Da greater in mass.
  • Figure 4f shows the yl7 ion, generated using IRMPD. This ion possesses its normal mass. The three ions in 4d, 4e and 4f indicate that the highlighted and underlined Ser274 is the site of covalent modification.
  • Figure 5a shows alignment of ybbR peptides with peptide sequences flanking the Ppant modified Ser (in box) in known PCPs and ACPs. The conserved Asp and Leu residues at the site of Ppant modification are highlighted. The position of helix II based on the NMR structures of TycC3-PCP and FrenN-ACP is also shown.
  • Figure 5b shows helical wheel plots of ybbR peptide and the helix II region of TycC3-PCP and FrenN- ACP starting with the Ppant modified Ser residue (underlined). Solvent exposed side of helix II in TycC3-PCP and FrenN-ACP are shaded.
  • Figure 6 depicts the kinetic analysis of Sfp catalyzed peptide ybbR13 modification by biotin CoA.
  • Figure 6a depicts the HPLC traces of the ybbR13 peptide labeling reaction with biotin CoA and Sfp added (trace 1) and the control reactions with either Sfp (trace 2) or biotin-CoA (trace 3) excluded from the reaction mixture.
  • Figure 6b depicts a Michaelis - Menten plot for the measurement of kinetic parameters of Sfp catalyzed ybbR13 labeling at saturating concentration of biotin- CoA (150 ⁇ M).
  • FIG. 7 is a schematic of the NMR structures of TycC3-PCP (PDB ID IDNY) and FrenN-ACP (PDB ID 1OR5). Ser45 in TycC-PCP and Ser39 in FrenN-ACP at the tip of helix II were posttranslationally modified by Sfp.
  • Figure 8 depicts the results of a CD spectra of the various peptides in 5 mM potassium phosphate, pH7.5 with 30% TFE.
  • Figure 8a summarizes the CD spectra analysis of the peptides ybbRl 1, ybbR12 and ybbR13 that were the substrates of Sfp.
  • Figure 8b summarizes the CD spectra analysis of the peptides ybbR3, ybbR8, ybbRl 4 and ybbRl 5, that were not the substrates of Sfp.
  • Figure 8c depicts the CD spectra analysis of peptides PksLl and TycC3 which include sequences that flank the Ppant modified Ser residue in PksL-PCP and TycC-PCP, respectively.
  • Figure 9 depicts the results of a Western blot analysis of biotin labeled ybbR fusions A-L as shown in the table above. Labeling reactions were carried out in cell lysates in which (1) both biotin - CoA and Sfp were added; (2) only biotin-CoA added; or (3) only Sfp was added.
  • Figure 10a is a schematic of an EGFP-ybbR145 construct including a ybbR peptide in the middle. The Ppant modified Ser residue in the ybbR peptide is underlined.
  • Figure 10b depicts a UV spectra of EGFP-ybbR145 labeled with tetramethylrhodamine or Texas Red by Sfp catalyzed ybbR labeling.
  • Figure 11 is a bar graph depicting the yield of the various labeled EGFP proteins including a ybbR peptide at the N-terminus, the C-terminus or in the middle. Yields of the protein labeling reactions were quantified by the binding of biotin labeled EGFP proteins to streptavidin beads.
  • EGFP proteins with an N-terminal ybbRl 2 tag (N- ybbR12-EGFP), an N-terminal ybbR13 tag (N-ybbR13-EGFP), an N-terminal six residue peptide DSLEFI (N-DSLEFI-EGFP), a C-terminal ybbRl 3 tag (C-ybbR13- EGFP) and an internal ybbRl 2 peptide at residue 145 (I-ybbR12-EGFP) were labeled with biotin by Sfp and biotin-CoA and immobilized on streptavidin beads.
  • Figure 12 is a schematic representation of the peptide labeling reaction and the selection scheme of the phage-displayed peptide libraries.
  • Figure 12a is a schematic of an Sfp or Acps-catalyzed reactions resulting in PCP, ACP or peptide tag labeling using small molecule-coA conjugates as the donor of the small molecule-Ppant group to a specific Serine residue in PCP, ACP or the peptide tags.
  • Figure 12b is a schematic depiction of structures of biotin-SS-CoA and biotin-CoA used in the selection and phage
  • Figure 12c is a schematic representation of the selection of the peptide substrates of Sfp and Acps from phage displayed peptide library.
  • Figure 13 is a bar graph depicting steady enrichment of the phage clones for the selection of the phage displayed peptide library by Sfp over five rounds of selection.
  • Figure 14 depicts an alignment of peptide sequences in the phage clones enriched by (a) Sfp- or (b) AcpS-catalyzed biotin labeling after the fifth round of selection.
  • the GDS sequence was not varied in the peptide library.
  • Figure 15 depicts the consensus sequences of the peptides displayed by the phage clones enriched by Sfp (a) and AcpS (b) catalyzed biotin labeling after five rounds of selection.
  • Ppant modified Ser is at position 3.
  • Figure 16 depicts the results of a phage ELISA from biotin labeling reactions with the phages displaying Sl and Al peptides.
  • Figure 17 depicts the results of a phage ELISA of the biotin labeling reactions with the phage clones enriched after the fifth round of selection.
  • Phage clones S4, S5 and S9 were from the Sfp selection and A2, A3 and A4 were from the Asps selection.
  • FIG. 18 depicts the results of an ELISA of N-terminal A- and S peptide- tagged EGFP labeled with biotin by Sfp- or Asps-catalyzed protein modification. N- terminal ybbR- or PCP-tagged EGFP were used as the controls.
  • Figure 19 depicts a CD spectra of the A and S peptides.
  • Figure 20 is a schematic representation of the helical wheel conformation of the ybbR13, Sl, S6 and Al peptide tags.
  • the Ppant modified Ser is underlined.
  • This invention is based, at least in part, on the identification of novel peptide substrates for enzymes having phosphopantetheinyl transferase activity such as for example, Sfp and Acps, and use of such substrates for site-specific labeling of both proteinaceous and non-proteinaceous molecules.
  • Sfp and Acps represent two classes of phosphopantetheinyl transferases (PPTases) that show differences both in their substrate specificity for the carrier protein domains and in their structures (Flugal et al, J. Biol. Chem., 275:959-968 (2000); Lambalot et al, Chem. Biol., 3:923-936 (1996)).
  • the Sfp class of PPTases are about 230 residues in size and the crystal structure of Sfp suggests it has a twofold symmetry with the N- and the C-terminal halves of the molecule adopting similar folds, with the active site of the enzyme at the interface (Hodneland et. al, Proc. Natl.
  • Acps is about 120 residues in length, about half the size of Sfp, and the crystal structures of Acps show that the enzyme assembles into trimers and the ACP and CoASH binding sites are formed at the interface between each monomer (Reuter et al, Embo. J., 18:6823-6831 (1999); Chirgadze et al, Embo. J., 19:5281-5287 (2000)).
  • Sfp exhibits a much broader substrate specificity than Acps in that Sfp can modify both PCP and ACP domains from nonribosomal peptides synthetases, polyketide synthases, and fatty acid synthases, while Acps modifies only ACP (Flugel et al, J. Biol. Chem., 275:959-968 (2000); Parris et al, Structure, 8:883-895 (2000); Mofid et al, J. Biol. Chem., 277:17023-17031 (2002)).
  • ACP and PCP carrier protein substrates of both kinds of PPTases adopt similar folds as four-helix bundle proteins with the serine residue to be modified by the Ppant prosthetic group at the top of the second ⁇ -helix, which has been shown to play an important role for interacting with Sfp and Acps (Hodneland et al, Proc. Natl. Acad. Sci. USA, 99:5048-5052 (2002); Chirgadze et al., Embo. J., 19:5281-5287 (2000); Quadri et al, Biochem., 37:1585-1595 (1998); Li et al, Biochem., 42:4648-4657 (2003)).
  • the present invention is based, at least in part, on the isolation and characterization of peptides, referred to as ybbR13, and the S and A series of peptides, specifically, S6 and Al, which are efficient substrates of phosphopantetheinyl transferases such as, for example, Sfp and Acps, respectively.
  • ybbR13 is an 11 amino acid residue peptide, which is a substrate of phosphopantetheinyl transferases such as, for example, S ⁇ .
  • the ybbR13 peptide has an amino acid sequence of DSJLEFIASKLA, set forth in SEQ ID NO:2, and was isolated from a phage displayed library of the B. subtilis genome.
  • a part of the sequence of the ybbR13 peptide is derived from a B. subtilis ORF, called ybbR, and it includes the DSL tripeptide sequence at the N-terminus, as shown in Figure 5 a, which is conserved in known substrates of Sfp, for example, PCP; but the ybbR peptide does not include the amino acid sequence, DxFFxxLGG (SEQ E) NO:3) at its N-terminus, which is found to be conserved in PCPs. Also described herein are modifications, truncations and variants of the ybbR13 peptide which can be used as substrates in phosphopantetheinylation reactions for site specific labeling of both proteinaceous and non-proteinaceous molecules.
  • Additional peptides described herein as efficient substrates for enzymes having phosphopantetheinyl transferase activity are the S series of peptides and the A series of peptides, designated as "S" or "A” based on their reactivity with Sfp or Acps, respectively.
  • Exemplary S series of peptides include, but are not limited to, S6, which is an efficient substrate for Sfp
  • exemplary A series of peptides include, but are not limited to, Al, which is an efficient substrate for Acps. Both S6 and Al peptides are 12 amino acid residues in length.
  • ybbR and S6 which are efficient substrates for Sfp and Al
  • Sfp and Al which is an efficient substrate for Acps
  • a pairing of ybbR or S6 with Sfp and Al with Acps can be used for site-specific labeling of two separate proteins, for example, either in cell lysates and/or on live cell surfaces. Such site- specific labeling of two separate proteins maybe carried out either sequentially or simultaneously.
  • the C-terminus of the ybbR13 peptide and not the N-terminus appears to be important for Sfp recognition.
  • the N- terminal extensions of the ybbR13 peptide e.g., ybbRl 1 and ybbR12
  • the ybbR13 peptide (SEQ ID NO: 2) had a K m of 122.8 ⁇ M for Sfp-catalyzed reaction, which is about thirty fold higher than that for PCP (4.1 ⁇ M); however, the k cat values for the ybbR peptide and PCP were similar at saturating concentrations of biotin - CoA. Sfp was able to transfer different types of small molecules from CoA to which they were conjugated, to the ybbR peptide. For example, Sfp catalyzed transfer of both fluorescein and biotin from CoA to the ybbR peptide at similar rates.
  • the ybbR13 peptide is shown herein, to be versatile with respect to its location, when used as a substrate in a phosphopantetheinylation reaction.
  • the ybbR13 peptide is recognized by Sfp, when fused either to the N- terminus, the C-terminus, or when located anywhere between the N- and the C-termini of various proteins.
  • the site specific labeling reaction of the ybbR tagged proteins can be carried out in cell lysates in just 10-20 minutes.
  • the amino acid residue that is modified by Sfp i.e., the Ppant modified Serine
  • a helix II which is important for Sfp recognition.
  • the corresponding helical wheel plots of the helix II region of the TycC3-PCP and FrenN-ACP with known NMR structures showed no significant similarities with the ybbR peptide with respect to the distribution or alignment of residues on a specific side of the helix.
  • the amphiphilic nature of the ybbR peptides appears to be unique to these substrates of phosphopantetheinyl transferases, as compared to the other known substrates.
  • the ybbR peptide could be inserted in the middle of a protein, e.g., EGFP, and still be labeled with various fluorophores using a Sfp catalyzed reaction.
  • a protein e.g., EGFP
  • Sfp catalyzed reaction it is contemplated that the Sfp-catalyzed posttranslational modification of the ybbR peptide can be used widely for versatile site-specific protein labeling and has many advantages over the existing methodologies.
  • the size of the ybbR13 peptide i.e., 11 residues
  • the size of PCP or ACP e.g., 75-80 residues
  • an 11 amino acid residue ybbR peptide e.g., ybbR13 or a variant thereof
  • ybbR13 an 11 amino acid residue
  • ybbR13 an 11 amino acid residue
  • ybbR13 an 11 amino acid residue
  • ybbR13 an 11 amino acid residue
  • a small molecule for example, from a donor molecule, e.g, CoA.
  • a ybbR sequence as short as 5 amino acid residues shown in SEQ ID NO:1 i.e., DSLXX, where X is any alpha helix favoring amino acid
  • DSLXX X is any alpha helix favoring amino acid
  • alpha-helical oligopeptide moieties the association of the ybbR peptides which adopt an ⁇ -helical conformation, either alone or when linked to a different amino acid sequence, with their ability to function as substrates in a phosphopantetheinylation reaction, these peptides are referred to herein as alpha-helical oligopeptide moieties.
  • the present invention also provides additional peptides such as, for example, the S series of peptides including, e.g., S6 and the A series of peptides including, e.g., Al, which can be fused to target proteins at either or both the N- and the C- terminals or anywhere between the N and C-termini, and be efficiently posttranslationally labeled with small molecules such as Ppant-fluorophores or Ppant-biotin by Sip and Acps, respectively, using various small molecule-CoA conjugates as the donors.
  • S6 and Al show significant orthogonality in reactivity with Sfp and Acps.
  • the catalytic efficiency (k cat /K m ) for Sfp-catalyzed S6 labeling is more than 440-fold higher than the Acp-catalyzed S6 labeling and conversely, the specific activity (k ca t /K m ) for Acps-catalyzed Al labeling is more than 30-fold higher than the Acps-catalyzed S6 labeling.
  • GFP proteins fused with the S6 tag or the Al tag are labeled with biotinyl-pantetheine with high efficiency only by Sfp or Acps, respectively.
  • the cross-labeling of S- tagged GFP by Acps and Al -tagged GFP by Sfp is at least 25-fold lower than the labeling of the S6 and Al tagged proteins by the respective PPTase, e.g., Sfp for the S6 peptide and Acps for the Al peptide.
  • a ybbR/Sfp or S6/S ⁇ pair in combination with a Al/Acps pair can be used for either simultaneous or sequential labeling of two target proteins with different small molecule probes, with very little or no cross labeling.
  • the S6 peptide appears to be a more efficient substrate of Sfp, relative to ybbR13, as demonstrated by the ratio of catalytic efficiencies (k cat /K m ) for Sfp- over Acps-catalyzed S6 labeling, which is 442 -fold compared to 28-fold for ybbR13 labeling.
  • the Examples herein demonstrate that the S6- or Al -tagged cell surface proteins including, but not limited to, for example, transferrin receptors, can be posttranslationally labeled with Sfp and Acps respectively. Due to the multiple negative charges on the ATP moiety of CoASH, CoA-conjugated small molecule probes are not membrane permeable.
  • CoA - fluorescent dye conjugate to penetrate the membrane is that the intracellular background fluorescence is very low after extracellular labeling of the cell with the conjugate. This feature allows high-contrast imaging of the intracellular trafficking of internalized cell surface receptors.
  • the present invention provides short peptide tags, e.g., ybbR13, S6 and Al, which can be used for the site-specific posttranslational labeling of target proteins in reactions catalyzed by a phosphopantetheinyl transferase such as, for example, Sfp or Acps. Additionally, a pairing of ybbR13/Sfp or S6/Sfp and Al/Acps can also be used for orthogonal site-specific labeling of two target proteins, e.g., in cell lysates or on the surface of live cells.
  • short peptide tags e.g., ybbR13, S6 and Al
  • a pairing of ybbR13/Sfp or S6/Sfp and Al/Acps can also be used for orthogonal site-specific labeling of two target proteins, e.g., in cell lysates or on the surface of live cells.
  • the small size of the peptide tags e.g., ybbR13, S6 and Al peptides compared to the full length PCP and ACP domains (80-100 residues), the versatility of the peptides for fusion to target proteins at N- or C-termmi, the structural diversities of the small molecule probes for Sfp- and Acps-catalyzed peptide modification, and the high efficiency and specificity of Sfp and Acps for the different peptides provide, a powerful protein labeling method that allows for specific orthogonal labeling of different target proteins on cell surfaces or in cell lysates.
  • this invention provides a method of labeling any proteinaceous or non-proteinaceous molecule (referred to herein as a "second agent”) by: (a) linking the molecule to an alpha-helical oligopeptide moiety having an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40 or SEQ ID NO:41, or a variant thereof; and (b) contacting the molecule with a desirable label (referred to herein as a "first agent") linked to a donor molecule (e.g., CoA or an analog thereof) via a linking group (e.g., the phosphopantetheine group or analog thereof of the CoA donor molecule), in the presence of a phosphopantetheinyl transferase such as, for example, Sfp or Acps, which transfers the label along with the linking group (i.e., "transferred moiety”) from the donor molecule to the alpha helical oligopeptide moiety, thereby
  • Phosphopantetheinyl transferases such as, for example, Sfp and Acps, have broad substrate specificities with respect to the first agents that may be transferred to an alpha-helical oligopeptide moiety, as described herein.
  • various first agents conjugated to CoA such as sugars, affinity probes such as biotin, glutathione, fluorescent probes such as fluorescein, Alexa Fluor dyes and redox probes such as porphyrin can be transferred to an alpha-helical oligopeptide moiety using a phosphopantetheinylation reaction.
  • this invention provides a method of labeling a proteinaceous molecule, such as, for example, a protein (i.e., a second agent), by linking an alpha-helical oligopeptide moiety to either the N-terminus of the protein (e.g., an alpha-helical oligopeptide moiety having an amino acid sequence set forth ejther in SEQ ID NO:1 or 2 or 40 or 41, or a variant thereof), the C-terminus of the protein (e.g., an alpha-helical oligopeptide moiety having an amino acid sequence set forth in SEQ ID NO:2 or 40 or 41, or a variant thereof), or anywhere between the N- and C- termini of the protein (e.g.
  • a proteinaceous molecule such as, for example, a protein (i.e., a second agent)
  • this invention provides methods for labeling a target protein comprising contacting a target protein with a first agent, and allowing the first agent to be conjugated to the target protein via an alpha-helical oligopeptide moiety, in the presence of a phosphopantetheinyl transferase, where the target protein is a fusion of the alpha-helical oligopeptide moiety and another polypeptide.
  • the target protein includes the alpha-helical oligopeptide moiety fused to the N-terminus of a polypeptide, where the alpha-helical oligopeptide moiety has an amino acid sequence set forth in SEQ ID NO:1 or a variant thereof.
  • the target protein includes the alpha-helical oligopeptide moiety fused to the N-terminus, the C-terminus, or anywhere between the N-terminus and the C-terminus of a polypeptide, where the alpha-helical oligopeptide moiety has an amino acid sequence set forth in SEQ ID NO:2 or a variant thereof.
  • the target protein includes at least two alpha-helical oligopeptide moieties, a first alpha- helical oligopeptide moiety having an amino acid sequence set forth in SEQ ID NO:1 or a variant thereof at the N-terminus of a polypeptide and one or more of an alpha-helical oligopeptide moiety having an amino acid sequence set forth in SEQ ID NO:2, or a variant thereof either the C-terminus of the polypeptide or located anywhere between the N- and the C-termini.
  • the target protein includes at least one alpha-helical oligopeptide moiety fused to the N-terminus, the C-terminus, or anywhere between the N-terminus and the C-terminus of a polypeptide, where the alpha-helical oligopeptide moiety has an amino acid sequence set forth in SEQ ID NO:40 or a variant thereof.
  • the target protein includes at least one alpha oligopeptide moiety fused to the N-terminus, the C-terminus, or anywhere between the N-terminus and the C-terminus of a target protein, where the alpha-oligopeptide moiety has an amino acid sequence set forth in SEQ ID NO:41 5 or a variant thereof.
  • a first target protein can include one or more alpha-helical oligopeptide moieties including the amino acid sequences set forth in SEQ ID NO:2 or SEQ ID NO:40, or a variant thereof
  • a second target protein can include an alpha-helical oligopeptide moiety including an amino acid sequence set forth in SEQ ID NO:41, or a variant thereof, either at the N-terminus, the C-terminus or anywhere between the N- and the C-termini.
  • the first target protein including at least one alpha helical oligopeptide moiety having an amino acid sequence selected from SEQ ID NO:2 or 40 can be labeled using an Sfp catalyzed reaction
  • the second target protein including at least one alpha helical oligopeptide moiety having an amino acid sequence set forth in SEQ ID NO:41 can be labeled using an Acps catalyzed reaction, either simultaneously or sequentially.
  • a first agent along with a linking group is transferred to a conserved serine residue on an alpha-helical . oligopeptide moiety via a phosphopantetheinylation reaction.
  • the first agent is attached to a linking group and a leaving group, and then is transferred with the linking group to the alpha-helical oligopeptide moiety via a phosphopantetheinylation reaction.
  • a schematic of such a reaction is shown below:
  • phosphopantetheinyl transferase and “protein having phosphopantetheinyl transferase activity,” as used herein, refer to any enzyme or a fragment thereof, which is capable of transferring a 4'-phosphopantetheine group from a donor molecule, such as, for example, coenzyme A (CoA) or an analog thereof, to a substrate such as, for example, an alpha-helical oligopeptide moiety.
  • a donor molecule such as, for example, coenzyme A (CoA) or an analog thereof
  • Phosphopantetheinyl transferases and proteins having phosphopantetheinyl transferase activity also include fragments, e.g., active fragments or fragments of a phosphopantetheinyl transferase or a protein having phosphopantetheinyl transferase activity, which are capable of transferring a transferred moiety (e.g., a first agent linked to a linking group) from a donor molecule such as, for example, M q -L-N, to a substrate.
  • a transferred moiety e.g., a first agent linked to a linking group
  • Phosphopantetheinyl transferases are enzymes which catalyze post- translational modification of carrier proteins associated with fatty acid synthetases (FASs), polyketide synthetases (PKSs) and non-ribosomal polypeptide synthetases (NPRs).
  • FOSs fatty acid synthetases
  • PKSs polyketide synthetases
  • NPRs non-ribosomal polypeptide synthetases
  • Phosphopantetheinyl transferases have been classified into three groups, based on sequence and structural similarity and substrate specificity. Members of the first group, for example, Acps of Escherichia coli, are about 120 amino acid residues long, function as homotrimers, and have fairly narrow substrate specificities limited to, for example, ACPs of type II FAS and PKS systems.
  • Phosphopantetheinyl transferases encompassed by this invention include both naturally occurring proteins having phosphopantetheinyl transferase activity including, but not limited to, Acps from E. Coli and Sfp from B. subtilis, fatty acid synthases (FAS) from S. cerevisiae, S. pombe, C. albacans, E. nidulans, and P. patulum, EntD from E. Coli, S.flexneri, S. typhimurium and S. austin, Psf-1 from B. pumilus, Gsp from B. brevis, Hetl from Anabaena sp., Lys5 from S.
  • FAS fatty acid synthases
  • a homolog of a phosphopantetheinyl transferase is intended to include phosphopantetheinyl transferases from a species other than the ones described, which are capable of phospopantetheinylating an alpha-helical oligopeptide moiety described herein.
  • a homolog of S ⁇ includes a phosphopantetheinyl transferase from a species other than B.
  • subtilis which is capable of phosphopantetheinylating an alpha-helical oligopeptide moiety including an amino acid sequence of DSLXX (SEQ ID NO:1) or DSLXXX (SEQ ID NO:39) or GDSLSWLLRLLN (SEQ ID NO:40) when linked to the N-terminus of any polypeptide, where X is an alpha-helix favoring amino acid.
  • a homolog of Sfp includes a phosphopantetheinyl transferase from a species other than B.
  • subtilis which is capable of phosphopantetheinylating an alpha-helical oligopeptide moiety including an amino acid sequence set forth in SEQ ID NO:2, or SEQ ID NO:40, or a variant thereof, independent of what it is linked to.
  • a homolog of Acps includes a phosphopantetheinyl transferase from a species other than E. CoIi, which is capable of phosphopantetheinylating an alpha-helical oligopeptide moiety including an amino acid sequence of GDSLDMLEWSLM (SEQ ID NO:41), or a variant thereof.
  • Homologs of phosphopantetheinyl transferases used in the methods described herein, for example, Sfp of B. subtilis, and Acps from E. CoIi, can vary in the degree of amino acid sequence homology or identity with the amino acid sequence of Sfp or Acps, so long as the homolog enzyme is capable of phosphopantetheinylating a substrate as described herein.
  • Homologs of Sfp and Acps within the scope of this invention can be encoded by a nucleic acid having any degree of nucleic acid sequence identity with a nucleic acid encoding B. subtilis Sfp (Grossman et al., J. Bacteriol., 175(19):6203-6211, 1993)).
  • Homology also termed herein "identity” refers to sequence similarity between two proteins (peptides) or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same nucleotide base or amino acid, then the molecules are homologous, or identical, at that position. A degree (or percentage) of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A degree or percentage of "identity" between amino acid sequences refers to amino acid sequence similarity wherein conserved amino acids are considered to be identical for the purpose of determining the degree or percentage of similarity. A conserved amino acid substitution is, e.g., substitution of one amino acid having a negative side chain for another amino acid having a negative side chain.
  • homologs of Sfp, Acps, or other members of the phosphopantetheine transferase superfamily have an overall amino acid sequence identity or similarity of at least about 50%, at least about 60%, at least about 70 %, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater than 99% with Sfp used in the methods described herein, wherein the homologs are capable of phosphopantetheinylating an alpha-helical oligopeptide moiety, as described herein.
  • Also encompassed by this invention are enzymes that are recombinantly or synthetically produced, and fragments thereof having phosphopantetheinyl transferase activity.
  • enzymes that are recombinantly or synthetically produced, and fragments thereof having phosphopantetheinyl transferase activity.
  • Examples of various members of the phosphopantetheinyl transferase family can be found, for example, in Lambalot et al., Chem. & Biol., 3:923-926, 1996 and in U.S. Patent No. 6,579,695, the entire contents of which are incorporated by reference herein.
  • Phosphopantetheinylation occurs by transfer of the 4'-phosphopantetheine (P- pant) prosthetic group from coenzyme A to a conserved serine residue in carrier proteins, which converts the carrier proteins from their inactive "apo” form to their active "holo” form. (Id. at Lambalot et al).
  • isolated and “purified,” as used in the context of a phosphopantetheinyl transferase or a protein having phosphopantetheinyl transferase activity, e.g., Sfp and Acps, refer to enzymes or fragments thereof that are substantially free of cellular material when purified, for example, from a cell which naturally expresses such an enzyme or fragment, or culture medium when purified from recombinant cells, or chemicals when produced synthetically.
  • Cells from which a phosphopantetheinyl transferase or a fragment thereof having phosphopantetheinyl transferase activity is isolated may either be prokaryotic or eukaryotic cells, including but not limited to, for example, yeast cells, plant cells, bacterial cells, insect cells and mammalian cells. Also encompassed by this disclosure are recombinant enzymes that may be "isolated” or “purified” from a cell that has been genetically engineered or modified to produce a phosphopantetheinyl transferase or fragment thereof having phosphopantetheinyl transferase activity.
  • a phosphopantetheinyl transferase used in the methods described herein is produced recombinantly or synthetically, it may not be necessary to purify the enzyme significantly.
  • a recombinantly produced phosphopantetheinyl transferase may have an activity identical to that of its natural counterpart, or it may have an activity which varies more or less from that of its natural counterpart.
  • a recombinantly produced phosphopantetheinyl transferase can vary in the efficiency of catalysis. It is possible to modify the amino acid sequence of the enzyme to, e.g., improve its efficiency, or to change its substrate specificity, or both.
  • Purification of a phosphopantetheinyl transferase or a protein or fragment thereof having phosphopantetheinyl transferase activity can be achieved using standard procedures well-known in the art. Exemplary procedures include, for example, affinity purification using a column containing a known substrate of a phosphopantetheinyl transferase enzyme such as, an apo-acyl carrier protein, or an alpha-helical oligopeptide moiety, as described herein.
  • a procedure used for purification of a phosphopantetheinyl transferase or a protein having phosphopantetheinyl transferase activity or a fragment thereof results in purification of a phosphopantetheinyl transferase or a protein or fragment having phosphopantetheinyl transferase activity to at least 50% purity, or at least 60% purity, or at least 70% purity, or at least 80% purity, or at least 90% purity, or at least 95% purity, or at least 96% purity, or at least 97% purity, or at least 98% purity, or at least 99% purity, or greater than 99% purity.
  • purification of a phosphopantetheinyl transferase or a protein having phosphopantetheinyl transferase activity or a fragment thereof results in an enrichment of phosphopantetheinyl transferase activity by at least about 500 fold, or at least 600 fold, or at least 700 fold, or at least 800 fold, or at least 900 fold, or at least 1000 fold, or at least 5000 fold, or at least 10,000 fold, or at least 50,000 fold, or at least 70,000 fold, or greater than 70,000 fold.
  • Exemplary methods of purification are described in, for example, U.S. Patent No. 6,579,695, the entire content of which is incorporated by reference herein.
  • a purified phosphopantetheinyl transferase or an enzyme having phosphopantetheinyl transferase activity has a specific activity of at least 100 mU/mg, or at least 200 mU/mg, or at least 250 mU/mg, or at least 300 mU/mg, or at least 400 mU/mg, or at least 500 mU/mg, or greater.
  • first agent refers to any entity, biological or synthetic in nature, which can be transferred from a donor molecule to an alpha-helical oligopeptide moiety.
  • a first agent is linked to a donor molecule such as, for example, coenzyme A, via a linking group, and is transferred along with the linking group or a part thereof, to an alpha-helical oligopeptide moiety.
  • the first agent is linked to the donor molecule via a covalent bond.
  • the first agent along with the linking group is transferred from the donor molecule to an alpha-helical oligopeptide moiety linked to a second agent using a phosphopantetheinylation reaction.
  • first agents include, but are not limited to, for example, small molecules moieties (e.g., drag moieties, toxins), protons, haptens, affinity probes, spectroscopic probes, radioactive probes, peptides, non-naturally occurring amino acids, nucleic acids, lipid molecules, radical generating molecules, singlet oxygen generating molecules, polymers, sugars (e.g., monosaccharides, disaccharides, polysaccharides, and other carbohydrate containing moieties), antibodies and antibody fragments, enzymes, enzyme substrates, chelating agents and receptor binding molecules.
  • spectroscopic probes includes, but is not limited to, moieties which can be detected using spectroscopic techniques.
  • spectroscopic probes include, but are not limited to, fluorophores (e.g., Fluorescein), chromophores (e.g., luminal, luciferase, luciferin, and aequorin), magnetic probes and contrast reagents (e.g., MRI contrast reagents).
  • fluorophores e.g., Fluorescein
  • chromophores e.g., luminal, luciferase, luciferin, and aequorin
  • magnetic probes e.g., MRI contrast reagents
  • contrast reagents e.g., MRI contrast reagents
  • Other examples of spectroscopic probes include, but are not limited to, phosphorescent probes and PET labels.
  • affinity probe includes, but is not limited to, moieties which can be used to bind to an affinity matrix, e.g., to enhance purification of molecules of the invention.
  • affinity probes include biotin and glutathione and analogs thereof.
  • biotin analogs include, for example, an N-ketone biotin analog, a ketone biotin analog, an N-azide biotin analog, an azide biotin analog, an N-acyl azide biotin analog, an NBD-GABA biotin analog, a 1,2-diamine biotin analog, an N-alkyne biotin analog and a tetrathiol biotin analog.
  • affinity matrix includes a matrix, such as agarose, controlled pore glass, or poly (styrenedivinyl) benzene to which an affinity ligand is attached.
  • the affinity ligand binds to the affinity probe and the contaminating molecules are not bound to the affinity ligand.
  • the molecule of the invention with the affinity probe can be eluted from the affinity matrix using known protocols.
  • antibody includes monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispeciflc antibodies (e.g., bispecifc antibodies), chimeric antibodies, CDR-grafted antibodies, humanized antibodies, human antibodies and antigen-binding fragments thereof, for example, an antibody light chain (VL), an antibody heavy chain (VH), a single chain antibody (scFv), a F(ab')2 fragment, a Fab fragment, an Fd fragment, an Fv fragment, and a single domain antibody fragment (DAb).
  • VL antibody light chain
  • VH antibody heavy chain
  • scFv single chain antibody
  • F(ab')2 fragment a single chain antibody
  • Fab fragment fragment
  • Fd fragment fragment
  • Fv fragment single domain antibody fragment
  • antibodies including, for example, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, fully human antibodies, antibodies produced in transgenic organisms, synthetic antibodies, single domain antibodies, antibodies with modified Fc regions, and camelid like antibodies are further described in, e.g., 6,331,415, 4,816,567; 5,225,539; 6,180,370; 5,693,762; 5,693,761; 5,585,089; 6,548,640; 5,859,205; 6,632,927; 6,407,213; 6,639,055; 5,885,793; 6,150,584; 5,770,429; 6,300,064; 6,846,634; 6,737,056; and 6,670,453, the entire contents of which are incorporated by reference herein.
  • receptor binding molecule includes an agonist, an antagonist or a ligand of a receptor, or analogs thereof.
  • a ligand may either be a natural ligand to which a receptor binds, or a molecule which is a functional analog of the natural ligand.
  • the functional analog may be a ligand with structural modifications, or may be a wholly unrelated molecule which has a molecular shape which interacts with the appropriate ligand binding determinants.
  • Ligands may serve as agonists or antagonists, see, e.g., Goodman, et al. (eds. 1990) Goodman & Gilman's: The Pharmacological Bases of Therapeutics, Pergamon Press, New York.
  • radioactive probes include radionuclides.
  • radionuclides include 123 Iodine, 125 Iodine, 131 Iodine, 105 Rhodium, 67 Gallium, 153 Sm, 177 Lu, 186 Re, 188 Re, 166 Ho, 67 Cu, 90 Y, l "indium, 18 Fluorine, or """Technetium (Tc99m).
  • Radionuclides for indirect labeling include, for example, 111 In and 90 Y.
  • the radionuclides used typically produce high energy ⁇ - or ⁇ -particles which have a short path length.
  • such radionuclides can be conjugated using methods described herein, to a molecule which would target them specifically to neoplastic cells, for example, thereby killing such cells.
  • chelating agents include, but are not limited to, 1- isothiocycmatobenzyl-3-methyldiothelene triaminepentaacetic acid (“MX-DTPA”) and cyclohexyl diethylenetriamine pentaacetic acid (“CHX-DTPA”) derivatives.
  • Other chelating agents include, for example, P-DOTA and EDTA derivatives, and EGTA.
  • first agents include non-naturally occurring amino acids.
  • non-naturally occurring amino acids include for glutamine (GIu) or glutamic acid residues: ⁇ -aminoadipate molecules; for tyrosine (Tyr) residues: phenylalanine (Phe), 4-carboxymethyl-Phe, pentafluorophenylalanine (PfPhe), 4- carboxymethyl-L-phenylalanine (cmPhe), 4-carboxydifluoromethyl-L-phenylalanine (F 2 cmPhe), 4-phosphonomethyl-phenylalanine (Pmp), (difluorophosphonomethyl) phenylalanine (F 2 Pmp), O-malonyl-L-tyrosine (malTyr or OMT), and fluoro-O- malonyltyrosine (FOMT); for proline residues: 2-azetidinecarboxylic acid or pipecolic acid (which have 6-membered,
  • enzymes or enzyme substrates as first agents.
  • examples of these include (enzyme (substrate)): Alkaline Phosphatase (4-Methylumbelliferyl phosphate Disodium salt; 3-Phenylumbelliferyl phosphate Hemipyridine salt); Aminopeptidase (L-Alanine-4-methyl-7-coumar- inylamide trifluoroacetate; Z-L-argimne-4-methyl-7-coumarinylamide hydrochloride; Z-glycyl-L- proline-4-methyl-7-coumarinylamide); Aminopeptidase B (L-Leucine-4-methyl-7- coumarinylamide hydrochloride); Aminopeptidase M (L-Phenylalanine 4-methyl-7- coumarinylamide trifluoroacetate); Butyrate esterase (4-Methylumbelliferyl butyrate); Cellulase (2-Chlor
  • drug moiety includes agents which would be beneficial to the subject.
  • Drug moieties may include, but are not limited to, anti-inflammatory, anticancer, cytotoxic, antiinfective (e.g., antifungal, antibacterial, anti-parasitic, anti- viral, etc.), and anesthetic agents.
  • drug moieties encompassed by this invention include anabolic agents, antacids, anti-asthmatic agents, anti-cholesterolemic and anti-lipid agents, anti-coagulants, anti-convulsants, anti-diarrheals, anti-emetics, anti-infective agents, anti-inflammatory agents, anti-manic agents, anti-nauseants, anti-neoplastic agents, anti-obesity agents, anti-pyretic and analgesic agents, anti-spasmodic agents; anti-thrombotic agents, anti-uricemic agents, anti-anginal agents, antihistamines, antitussives, appetite suppressants, cerebral dilators, coronary dilators, decongestants, diuretics, erythropoietic agents, expectorants, gastrointestinal sedatives, hyperglycemic agents, hypnotics, hypoglycemic agents, laxatives, mucolytic agents, neuromuscular drugs, peripheral vaso
  • drug moieties include: anti-neoplasties such as androgen inhibitors, antimetabolites, cytotoxic agents, and immunomodulators; anti-tussives such as dextromethorphan, dextromethorphan hydrobromide, noscapine, carbetapentane citrate, and chlorphedianol hydrochloride; antihistamines such as chlorpheniramine maleate, phenindamine tartrate, pyrilamine maleate, doxylamine succinate, and phenyltoloxamine citrate; decongestants such as phenylephrine hydrochloride, phenylpropanolamine hydrochloride, pseudoephedrine hydrochloride, and ephedrine; various alkaloids such as codeine phosphate, codeine sulfate and morphine; antiarrhythmics such as N-acetylprocainamide; antipyretics and analgesics such as acetamin
  • drug moieties include analgesics, such as nonsteroidal antiinflammatory drugs, opiate agonists and salicylates; antihistamines, such as H 1 -blockers and H 2 -blockers; anti-infective agents, such as anthelmintics, antianaerobics, antibiotics, aminoglycoside antibiotics, antifungal antibiotics, cephalosporin antibiotics,
  • analgesics such as nonsteroidal antiinflammatory drugs, opiate agonists and salicylates
  • antihistamines such as H 1 -blockers and H 2 -blockers
  • anti-infective agents such as anthelmintics, antianaerobics, antibiotics, aminoglycoside antibiotics, antifungal antibiotics, cephalosporin antibiotics,
  • antineoplastic agents such as alkylating agents, nitrogen mustard aklylating agents, nitrosourea alkylating agents, antimetabolites, purine analog antimetabolites, pyrimidine analog antimetabolites, hormonal antineoplastics, natural antineoplastics, antibiotic natural antineoplastics, and vinca alkaloid natural antineoplastics
  • autonomic agents such as anticholinergics, antimuscarinic anticholinergics, ergot alkaloids, parasympathomimetics, cholinergic agonist parasympathomimetics, cholinesterase
  • cardiovascular agents such as antianginals, ⁇ -blocker antianginals, calcium-channel
  • blocker antianginals nitrate antianginals, antiarrhythmics, cardiac glycoside antiarrhythmics, class I antiarrhythmics, class II antiarrhythmics, class III antiarrhythmics, class IV antiarrhythmics, antihypertensive agents, .alpha. -blocker antihypertensives, angiotensin-converting enzyme inhibitor (ACE inhibitor)
  • ACE inhibitor angiotensin-converting enzyme inhibitor
  • antihypertensives such as antihypertensives, ⁇ -blocker antihypertensives, calcium-channel blocker antihypertensives, central-acting adrenergic antihypertensives, diuretic antihypertensive agents, peripheral vasodilator antihypertensives, antilipemics, bile acid esequestrant antilipemics, HMG-CoA reductase inhibitor antilipemics, inotropes, cardiac glycoside inotropes, and thrombolytic agents; dermatological agents, such as antihistamines, antiinflammatory agents, corticosteroid anti-inflammatory agents, antipruritics/local anesthetics, topical anti-infectives, antifungal topical anti-infectives, antiviral topical anti-infectives, and topical antineoplastics; electrolytic and renal agents, such as acidifying agents, alkalinizing agents, diuretics, carbonic anhydrase inhibitor diuretics, loop di
  • glaucoma agents ⁇ -blocker anti-gluacoma agents, miotic anti-glaucoma agents,
  • mydriatics mydriatics, adrenergic agonist mydriatics, antimuscarinic mydriatics, ophthalmic anesthetics, ophthalmic anti-infectives, ophthalmic aminoglycoside anti-infectives, ophthalmic macrolide anti-infectives, ophthalmic quinolone anti-infectives, ophthalmic sulfonamide anti-infectives, ophthalmic tetracycline anti-infectives, ophthalmic antiinflammatory agents, ophthalmic corticosteroid anti-inflammatory agents, and ophthalmic nonsteroidal anti-inflammatory drugs (NSAIDs); psychotropic agents, such as antidepressants, heterocyclic antidepressants, monoamine oxidase inhibitors (MAOIs), selective serotonin reuptake inhibitors (SSRIs), tricyclic antidepressants, antimanics, antipsychotics, phenothiazine antipsychotics, an
  • cytotoxic drugs for example, those which are used for cancer therapy, e.g., anticancer agents.
  • cytotoxin means any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit or destroy a cell or malignancy.
  • cytotoxins include, but are not limited to, radionuclides, biotoxins, enzymatically active toxins, cytostatic or cytotoxic therapeutic agents, prodrugs, immunologically active ligands and biological response modifiers such as cytokines. Any cytotoxin that acts to retard or slow the growth of immunoreactive cells or malignant cells is within the scope of the present disclosure.
  • cytostatic agents include, in general, cytostatic agents, alkylating agents, antimetabolites, antiproliferative agents, tubulin binding agents, hormones and hormone antagonists, and the like.
  • cytostatics that are compatible with the present invention include alkylating substances, such as mechlorethamine, triethylenephosphoramide, cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan or triaziquone, also nitrosourea compounds, such as carmustine, lomustine, or semustine.
  • cytotoxic agents include, for example, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, and the podophyllotoxins.
  • adriamycin carminomycin, daunorubicin (daunomycin), doxorubicin, aminopterin, methotrexate, methopterin, mithramycin, streptonigrin, dichloromethotrexate, mitomycin C, actinomycin-D, porfiromycin, 5-fluorouracil, floxuridine, florafur, 6-mercaptopurine, cytarabine, cytosine arabinoside, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine and the like.
  • Still other cytotoxins include taxol, taxane, cytochalasin B, gramicidin D, ethidium bromide, emetine, tenoposide, colchicin, dihydroxy anthracin dione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Hormones and hormone antagonists such as corticosteroids, e.g. prednisone, progestins, e.g. hydroxyprogesterone or medroprogesterone, estrogens, e.g. diethylstilbestrol, antiestrogens, e.g. tamoxifen, androgens, e.g. testosterone, and aromatase inhibitors, e.g. aminogluthetimide may also be used as first agents.
  • corticosteroids e.g. prednisone
  • progestins
  • drug moieties include, for example, anticancer agents such as angiogenesis inhibitors (e.g., Angiostatin Kl-3, DL- ⁇ -Difluoromethyl-ornithine, Endostatin, Fumagillin, Genistein, Minocycline, Staurosporine, and ( ⁇ )-Thalidomide); DNA-intercalator or cross-linkers (e.g., Bleomycin, Carboplatin, Carmustine, Chlorambucil, Cyclophosphamide, cis-Diammineplatinum(II) dichloride (Cisplatin), Melphalan, Mitoxantrone, and Oxaliplatin); DNA synthesis inhibitors (e.g., ( ⁇ )- Amethopterin (Methotrexate), 3-Amino-l,2,4-benzotriazine 1,4-dioxide, Aminopterin, Cytosine ⁇ -D-arabinofuranoside, 5-Flu
  • Leuprolide Leuprorelin
  • Luteinizing Hormone-Releasing Hormone Pifithrin- ⁇
  • Examples of drug moieties further include doxorubicin, etoposide, taxane, paclitaxel, fluorouracyl, mitomycin, camptothecin, gemcitabine, geldanamycin, epothilone, cephalostatin, tubulin inhibitors, a vinca alkaloid, proteasome inhibitors, neocarzinostatin, calicheamicin, maytansinoids, (RS)-cyclophosophamide, 6- mercaptopurines, auristatin E, daunorubicin, and derivatives or analogs thereof.
  • the drug moieties may be attached to the linking group through any atom which allows the resulting molecule and/or drug moiety to perform its intended function.
  • the drug moieties also include pharmaceutically acceptable prodrugs, salts, esters, amides, and ethers of the drug moieties described herein. Derivatives include modifications to drugs identified herein which may improve or not significantly reduce a particular drug's desired therapeutic activity.
  • the term "second agent" includes any moiety, proteinaceous or non- proteinaceous, which is attached to a first agent through a linking group using methods described herein.
  • the second agent is a proteinaceous molecule such as, for example, a polypeptide, an antibody, a fusion protein or a hormone. Examples of proteinaceous second agents include, but are not limited to, interleukins 1
  • LHRH hormone releasing hormone
  • analogues gonadatropin releasing hormone
  • GnRH transforming growth factor- ⁇
  • FGF fibroblast growth factor
  • necrosis factor- ⁇ & ⁇ necrosis factor- ⁇ & ⁇
  • NGF nerve growth factor
  • GHRF growth factor releasing factor
  • EGF epidermal growth factor
  • FGFHF fibroblast growth factor homologous factor
  • HGF hepatocyte growth factor
  • IGF insulin growth factor
  • PDGF platelet-derived growth factor
  • IIF-2 invasion inhibiting factor-2
  • BMP 1-7 morphogenetic proteins 1-7
  • somatostatin thymosin- ⁇ -1
  • ⁇ -globulin ⁇ -globulin
  • superoxide dismutase SOD
  • complement factors hormones such as, for example, insulin, interferons or cytokines and other bioactive peptidic compounds, such as hGH, tPA, calcitonin, ANF, EPO, insulin
  • antibodies e.g., human anti-TAC antibody
  • recombinant beta-glucan bovine immunoglobulin
  • bovine superoxide dismutase recombinant hirudin (r-Hir), HIV-I immunogen
  • r-hGH human hemoglobin
  • r-Hb recombinant human mecasermin
  • r- IGF-I recombinant interferon beta-la
  • lenograstim G-CSF
  • olanzapine recombinant thyroid stimulating hormone (r-TSH); and topotecan.
  • any protein can be labeled using methods of the invention by simply fusing the protein to an alpha-helical oligopeptide moiety, as described herein.
  • the second agent is linked to an alpha-helical oligopeptide moiety, as described above.
  • the second agent may be linked through its N-terminus to an alpha-helical oligopeptide moiety (e.g., an alpha-helical oligopeptide moiety having an amino acid sequence set forth in SEQ 3D NO: 1 or 2 or 40 or 41 or a variant thereof).
  • the second agent when the second agent is proteinaceous, the second agent may be linked to one or more alpha-helical oligopeptide moieties having an amino acid sequence set forth in SEQ H) NO:2 or SEQ ID NO:40 or 41 or a variant thereof at its N-terminus, the C-terminus or anywhere in between the N-terminus and the C-terminus.
  • the second agent may be a non-proteinaceous moiety such as, for example, a nucleic acid molecule or a derivative thereof, a lipid molecule or a derivative thereof, or a carbohydrate or a derivative thereof.
  • a second agent may also be a small molecule moiety, as described herein.
  • second agents include, for example, an affinity probe, a spectroscopic probe, a radioactive probe, a radical generating molecule, a singlet oxygen generating molecule, a polymer, a hapten, a chelating agent or a receptor binding molecule, which may be linked to at least one alpha-helical oligopeptide moiety, such as, for example, an alpha helical moiety having an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40 or SEQ ID NO:41, or a variant thereof.
  • the second agent includes at least two portions, a proteinaceous portion and a non-proteinaceous portions.
  • the second agents may further comprise one or more epitope tags such as, for example, a flag tag, a GST tag, a His tag, an HA-tag.
  • epitope tags such as, for example, a flag tag, a GST tag, a His tag, an HA-tag.
  • alpha-helical oligopeptide moiety refers to an oligopeptide or variant thereof which is capable of forming an alpha helical structure either when linked to another peptide (for example, in case of the amino acid sequence set forth in SEQ ID NO:1, or a variant thereof); or is capable of forming an alpha helical structure independent of the second agent (for example, the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40 or SEQ ID NO:41 or a variant thereof).
  • alpha- helical oligopeptide moiety includes peptides which are capable of forming an alpha- helical structure in solution such as, for example, peptides including an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40, or a variant thereof, and those peptides which are capable of forming an alpha-helical structure upon binding to an enzyme having phosphopantetheinyl transferase activity such as, for example, the peptide having an amino acid sequence set forth in SEQ ID NO:41, or a variant thereof.
  • the alpha-helical oligopeptide moiety is linked to a second agent and is capable of accepting a first agent-linking group conjugate via a phosphopantetheinylation reaction.
  • the alpha-helical oligopeptide moiety includes the amino acid sequence DSL and at least two other alpha- helix favoring amino acids.
  • the alpha-helical oligopeptide moiety includes the amino acid sequence DSL and at least three other alpha helix favoring amino acids.
  • an alpha-helical oligopeptide moiety is capable of forming an alpha-helical structure independent of the second agent to which it is linked.
  • the alpha-helical oligopeptide moiety includes the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40 or SEQ ID NO:41, or a variant thereof, which can form an alpha-helical structure independently of the second agent, to which it is linked, either in solution or upon binding to an enzyme having phosphopantetheinyl transferase activity.
  • Alpha- helical oligopeptide moieties may either include D-amino acids, L-amino acids, or a combination thereof.
  • nucleic acid molecules encoding the alpha-helical oligopeptide moieties described herein.
  • Variants of alpha helical oligopeptide moieties include peptides which include one or more amino acid mimetics and are capable of accepting a first agent-linking group conjugate in a phosphopantetheinylation reaption.
  • An "amino acid mimetic” refers to a moiety, other than a naturally occurring amino acid, that conformationally and functionally serves as a substitute for a particular amino acid in a peptide compound without adversely interfering with the function of the peptide.
  • “Alpha-helix favoring amino acids” are those amino acids which favor formation of an alpha helical structure when present in a peptide.
  • alpha helix favoring amino acids include, for example, Ala, GIu, Leu and Met.
  • Alpha-helix favoring amino acids as used herein, specifically exclude those amino acids which either prevent or hinder the formation of an alpha helical structure when included in a peptide. Examples of such amino acids include Pro, GIy, Tyr and Ser.
  • linking groups are typically linked to a first agent, which along with the linking group is transferred to an alpha-helical oligopeptide moiety.
  • linking group may be linked to I 5 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 first agents.
  • Linking groups and first agents are selected such that they are capable of being transferred to the alpha-helical oligopeptide moieties encompassed by this disclosure, through a phosphopantetheinylation reaction.
  • the linking group is phosphopantetheine or a derivative thereof, hi a further embodiment, the linking group (shown linked to a first agent M) is of the formula:
  • each R is M, optionally substituted alkyl, halogen, alkoxy, hydroxy, or hydrogen independently selected for each occurence;
  • U 1 is oxygen, sulfur or N-M
  • U 2 is O-M, S-M, CR 2 -M, or NRM; and each M is an independently selected first agent for each occurrence.
  • the linking group (shown linked to a first agent M) is of the formula: wherein each R is M, optionally substituted alkyl (e.g., CM 3 , etc.), halogen, alkoxy, hydroxy, or hydrogen independently selected for each occurence;
  • U 1 is oxygen, sulfur or N-M
  • U 2 is O-M, S-M, CR 2 -M, or NRM; and each M is an independently selected first agent for each occurrence.
  • the linking group linked to a first agent is O-M, S-M, CR 2 -M, or NRM; and each M is an independently selected first agent for each occurrence.
  • each R is M, optionally substituted alkyl (e.g., CM 3 , etc.), halogen, alkoxy, hydroxy, or hydrogen independently selected for each occurence;
  • U 1 and U 3 are each independently oxygen, sulfur or N-R;
  • U 2 is oxygen, sulfur, CR 2 , or N-M
  • U 4 is NR 2 , CR 3 , S-R or O-R; and each M is an independently selected first agent for each occurrence.
  • Examples of linking groups linked to first agents ⁇ e.g., M q -L) include moieties of the formula (V):
  • M q -L is of the formula (VI):
  • each M is an independently selected first agent for each occurrence.
  • the term "leaving group,” as used herein, includes groups that when linked to the linking group and first agent are capable of departing in a phosphopantetheinylation reaction, resulting in the transfer of the linking group and first agent to the alpha-helical oligopeptide moiety. Examples of leaving groups include phosphorylated nucleotides such as 3', 5'-ADP.
  • compositions including one or more molecules produced by the methods described herein.
  • pharmaceutical compositions further include a pharmaceutically acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration into a subject.
  • Methods of the invention can be used, for example, for labeling two proteins with two separate compounds.
  • two proteins present in a cell lysate can be labeled using the methods of the invention using a Sfp catalyzed reaction for labeling one protein and an Acps catalyzed reaction for labeling a second protein.
  • one protein can be linked with one or more alpha-helical oligopeptide moieties including an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40
  • the second protein can be linked with an alpha- helical oligopeptide moiety which includes the amino acid sequence set forth in SEQ ID NO:41.
  • the cell lysate including both proteins can be contacted first with the Sfp enzyme or a fragment thereof, having phosphopantetheinyl transferase activity in the presence of a first compound, thereby labeling the protein linked to the alpha-helical oligopeptide moiety including the amino acid sequence set forth in either SEQ ID NO:2 or 40.
  • the cell lysate can be contacted with the Acps enzyme or a fragment thereof, having phosphopantetheinyl transferase activity in the presence of a second compound, thereby labeling the protein linked to the alpha-helical oligopeptide moiety including the amino acid sequence set forth in SEQ ID NO:41.
  • an Acps catalyzed reaction can be carried out before the Sfp catalyzed reaction.
  • Such a method can also be used for labeling one or more proteins on the surface of live cells.
  • a method of delivering a second agent such as, for example, a protein, to a desired location in a subject.
  • a method of delivering a second agent to a desired location within a subject includes for example, administering to a subject a molecule of formula (I), where the first agent is capable of delivering the second agent to the desired location within the subject, where the molecule of formula (I) is:
  • L is a linking group
  • Y is an alpha-helical oligopeptide moiety
  • Z is a second agent.
  • molecules made using methods described herein can be used for disease detection and/or diagnosis.
  • a second agent is molecule which recognizes a tumor specific antigen (e.g., an antibody which binds a tumor specific antigen).
  • a tumor specific antigen e.g., an antibody which binds a tumor specific antigen
  • an antibody or a fragment thereof which specifically binds a tumor specific antigen for example, can be linked to a first agent (e.g., a suitable tag for fluorescent imaging) using methods described herein and administered to a subject, where detection of the fluorescent first agent is indicative of the presence of the tumor in the subject.
  • molecules made using methods described herein can be used for simultaneous administration of two or more agents to a subject, for example, for treatment of a disorder or a disease, where combination of the two or more agents is effective for treatment.
  • a first agent can be a hormone (e.g., insulin) used for the treatment of diabetes
  • a second agent can be a drug which is commonly used in conjunction with insulin for the treatment of diabetes. Therefore, insulin conjugated with a second agent can be generated using methods described herein and used in the treatment and/or management of diabetes.
  • Tbis invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference.
  • reaction mixtures were stirred at room temperature for about one hour in dark followed by purification with preparative HPLC on a reverse-phase Cl 8 column with a gradient of 0-50% acetonitrile in 0.1% TFA/water for 30 minutes.
  • the purified compound was lyophilized and the identity was confirmed by MALDI-TOF (negative mode): [M-H] " , fluorescein - CoA, calculated 1193.2, observed 1192.8; tetramethylrhodamine - CoA, calculated 1248.3, observed 1247.8; and Texas Red - CoA, calculated 1509.3, observed 1509.2.
  • Peptides were synthesized by the Biopolymer Laboratory of the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School.
  • Biotin ⁇ SS-CoA was synthesized with the following procedure. To a solution of biotin HPDP (PIERCE) (10.8 mg, 0.020 mmol) in 500 ⁇ L DMSO, coenzyme A lithium salt (SIGMA) (16.5 mg, 0.022 mmol) in 2 ml sodium phosphate, 100 mM, pH 7.0, was added and the reaction mixture was stirred at room temperature for one hour.
  • biotin HPDP PIERCE
  • SIGMA coenzyme A lithium salt
  • reaction mixture was then purified by preparative HPLC on a reversed-phase Cl 8 column with a gradient of 0-60% acetonitrile in 0.1% TF A/water over 35 minutes.
  • the purified compound was lyophilized and its identity was confirmed by LC-MS (negative mode) [M-H " ]: 1194.72, calculated for C 40 H 67 N n O 19 P 3 S 3 " : 1194.31.
  • Synthesis of Alexa 488-CoA and Texas red-CoA was previously reported (Yin et ah, Proc. Natl. Acad. Sci. USA 102:15815-15820 (2005)).
  • the phagemid DNA was transformed into XLl -Blue competent cells (STRAT AGENE) by electroporation and the cells were plated on LB agarose plates supplemented with 2% (w/v) glucose and 100 ⁇ g/mL ampicillin. After incubation at 37°C overnight, colonies on the plates were picked and the phagemid DNA was extracted by a Qiagen Plasmid Maxi kit (QIAGEN).
  • Phagemids including various lengths of the ybbR gene were isolated from the selected phage clones by a DNA miniprep ldt (QIAGEN).
  • the B. suhtilis genomic DNA fragments were sequenced using primers Junl3 (5'- ACT TTA TGC TTC CGG CTC GTA TGT) (SEQ ID NO:7) and Junl4 (5'- AAT CAA AAT CAC CGG AAC CAG AGC) (SEQ ID NO: 8) and the ybbR gene fragment (from clone JY529) was subcloned into the ⁇ ET-21b plasmid (NOVAGEN) using the EcoRI and HindIII restriction sites for enabling the expression of JY529 as a fusion protein with a C-terminal 6 ⁇ Histidine tag (JY529-His).
  • Primer Junl67 also inserted a HindIII site in between the ybbR12 coding sequence and the EGFP gene.
  • the PCR fragment was subsequently cloned into pET22b vector (NOVAGEN) between the Ndel and Xhol sites resulting in the pET22b-ybbR12-EGFP construct for the expression of EGFP with ybbR12 fused to the N-terminus and a 6xHistidine tag at the C-terminus.
  • GST and MBP genes were amplified using primer pairs Jun58 (5'- GAA ACA GTA TTC AAG CTT CCT ATA CTA GGT TAT TGG) (SEQ ID NO:13) / Jun60 (5'- GGA TCC ACG CGG CTC GAG ATC CGA TTT TGG) (SEQ ID NO: 14 ) and Jun ⁇ l (5'- GGA CCA TAG CAT AAG CTT ATC GAA GAA GGT AAA CTG G) (SEQ ID NO:15) / Jun63 (5'- CAG GTC GAC TCT CTC GAG TCC GAA TTC TGA AAT CC) (SEQ ID NO: 16), respectively, which introduced Hindi ⁇ and Xhol sites at the 5' and 3'- ends, respectively, of the PCR fragments.
  • the amplified GST and MBP genes were subsequently cloned into ⁇ ET22b-ybbR12-EGFP between the HindIII and Xhol sites, resulting in the constructs ⁇ ET22b-ybbR12-GST and pET22b-ybbR12-MBP for the expression of N-terminal ybbR tagged ybbR-GST and ybbR12-GST fusions, respectively.
  • primers Junl69 (5'- GAA TCC AGC CCC CAT ATG GAT TCT CTT GAA TTT ATT GCT AGT AAA CTG GCG AAG CTT GTG AGC AAG GGC GAG G) (SEQ ID NO: 17) and Jun57 were used to PCR amplify EGFP genes for the construction of expression vector pET-ybbR13-EGFP which expresses the N- terminal ybbR13-EGFP fusion.
  • the amplified PCR fragment further included the Ndel and BamHI sites at the 5' and 3 '-ends respectively for the cloning of the fragment into pET14b (NOVAGEN), resulting in the plasmid ⁇ ET14b-EGFP-ybbR12.
  • EGFP including ybbR12 fused to the C-terminal and a 6 ⁇ Histidine tag at the N-terminal was subsequently expressed.
  • Primer Junl73 also introduced an Xhol site at the end of EGFP gene immediately before the ybbR12 coding sequence.
  • the Acps gene was amplified from pPDJ (Yin et ah, J. Am. Chem. Soc, 126:3006-3007(2004)) with primers 5'-TCT GGT CAT ATG GCA ATA TTA GGT TTA GGC ACG G (SEQ ID NO:44) and 5'- TCA AGT CTC GAG TTA ACT TTC AAT AAT TAC CGT GGC A (SEQ ID NO:45). The resulting PCR product was digested with Ndel and Xhol and cloned into corresponding restriction sites of the pET28b vector (NOVAGEN).
  • ACPS protein was overexpressed in BL21 DE3 star cells (DSrVITROGEN) at 3O 0 C for 6 hours.
  • the N-terminal His-tagged recombinant ACPS protein was purified with standard Ni-NTA purification procedures and dialyzed into buffer containing 100 mM Bis-Tris propane (pH 6.0), 500 niM NaCl and 10% glycerol. Expression of Sfp was carried out, as previously reported (Yin et ah, Nature Protocols 1:280-285 (2006)).
  • E. coli XLl-Blue cells were transformed with pComb vectors, and shaken at 37°C in 2X yeast/tryptone broth and 100 ⁇ g/ml ampicillin.
  • helper phage VCSM13 was added to a final concentration of 1.5 X 10 s cfu/ml, and incubated at 37°C for 1 hour without shaking.
  • the cells were pelleted and resuspended in 2X YT, 100 ⁇ M isopropyl- D-thiogalactoside (IPTG), 100 ⁇ g/ml ampicillin, and 50 ⁇ g/ml kanamycin, and shaken for 14 h at room temperature. The following day, cells were pelleted and phage particles in the supernatant were precipitated using polyethylene glycol, followed by resuspension in TBS (25 mM Tris-HCl, pH 7.4/140 mM NaCl/2.5 mM KCl). Phage titrations were performed with E. coli XLl -blue using standard procedures. VI. Phage selection.
  • the number of the input phage particles, concentration of enzymes and biotin-SS-CoA and the reaction time were decreased step by step and eventually for the fifth round of selection, only 10 10 phage particles were incubated with 0.08 ⁇ M enzyme and 1 ⁇ M biotin-SS-CoA for 7 minutes at 30°C. Control reactions were also run in parallel without the addition of enzymes or biotin-SS-CoA.
  • reaction mixtures were added to 250 ⁇ L of 20% (w/v) polyethylene glycol 8000 with 2.5 M NaCl followed by 10 minute incubation on ice.
  • the phage particles in the reaction mixture were then precipitated by centrifugation at 4 0 C at a speed of 13,000 revolutions per minute (rpm).
  • the phage pellet was subsequently resuspended in 1 mL TBS supplemented with 1% (w/v) BSA and distributed in 100 ⁇ L aliquots to the wells of streptavidin coated 96 well plates (PIERCE).
  • the plates were allowed to incubate at room temperature for one hour before the supernatant was discarded and each well was washed 30 times with 0.05% (v/v) Tween 20, 0.05% (v/v) Triton X-100 in TBS and 30 times with TBS, each time with 200 ⁇ L of solution. After washing, phages bound to the streptavidin surface were eluted by adding 100 ⁇ L 20 mM dithiothreitol in TBS to each well to induce the cleavage of the disulfide bond that links the biotin group with Ppant.
  • Eluted phage particles were combined, added to 10 mL of log phase XLl -Blue cells and shaken at 37 0 C for one hour to infect the cells. The cells were then plated on LB agarose plates supplemented with 2% (w/v) glucose and 100 ⁇ g/mL ampicillin. After incubation at 37°C for overnight, colonies on the plates were scratched and the phagemid DNA was extracted by a Qiagen Plasmid Maxi kit. The phagemid DNA was then used for the next round of phage production and selection. Also phage particles eluted from the wells loaded with either the selection and the control reactions were titered in order to count the number of phage particles selected by each round. After the fifth round of selection, phage clones were sequenced using the primer Junl3 (5'- ACT TTA TGC TTC CGG CTC GTA TGT) (SEQ ID NO:7).
  • Cross-plate series dilution of the reaction mixtures was carried out by transferring 20 ⁇ L of the reaction mixture in 1% BSA to the wells in the column using a multichannel pipetor. The streptavidin plate was then incubated at room temperature for 60 minutes and washed five times with 0.05% (v/v) Tween 20, 0.05% (v/v) Triton X-100 in TBS, followed by washing five times with TBS, each time with 200 ⁇ L of solution. 100 ⁇ L 1/5000 diluted anti-M13 HRP conjugate (GE HEALTHCARE) in 1% BSA in TBS was added to each well and incubated at room temperature for another hour.
  • GE HEALTHCARE diluted anti-M13 HRP conjugate
  • the plate was again washed five times with 0.05% (v/v) Tween 20, 0.05% (v/v) Triton X-100 in TBS and five times with TBS.
  • the plate was subsequently developed by adding 100 ⁇ L TMB peroxidase substrate and hydrogen peroxide mixture (PIERCE) to each well and incubated at room temperature for five minutes before being photographed.
  • PIERCE hydrogen peroxide mixture
  • JY529-His (1.4 mM) was combined with Sfp (4 ⁇ M) and CoA (1 mM) to the parenthetically indicated final concentrations in 100 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 and 1 mM TCEP, and incubated at 3O 0 C for 4 hours to allow for phosphopantetheinylation.
  • the mixture was directly injected onto a 4.6 x 150 mm Jupiter C4 reversed phase column and eluted in a linear gradient of 15 to 55% CH 3 CN over 40 minutes at a flow rate of 1 mL min "1 .
  • the absorbance was monitored at 220 nm and two peaks eluting at 26 and 29 minutes were collected.
  • the fractions were lyophilized and re-suspended in 200 ⁇ L of 50:50 H 2 OiCH 3 CN with 0.1% formic acid and directly infused into a custom built quadrupole Fourier-Transform mass spectrometer (Q-FTMS) via a Nanomate nanoelectrospray robot. After the ions were passed through a resistively heated metal capillary/ they were externally accumulated in an octupole for 500 ms (Senko et al., J. Am. Mass. Spectrom, 8:970-976, 1997).
  • the ions were subsequently shuttled to the analyzer cell through a quadrupole that can act as a simple ion guide or a filter for selected m/z windows.
  • the data were fit using a least squares algorithm THRASH (Horn et al., J. Am. Soc. Mass Spectrom, 11 : 320-332, 2000) and the monoisotopic and most abundant isotopic peaks were determined. Tandem mass spectrometry was accomplished by first using the quadrupole to mass select specific charge states prior to subjecting the ions to octupole collisionally activated dissociation (OCAD), infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD).
  • OFC octupole collisionally activated dissociation
  • IRMPD infrared multiphoton dissociation
  • ECD electron capture dissociation
  • reactions were then quenched by adding 30 ⁇ L 4% trifluoroacetic acid (TFA) and analyzed using analytical HPLC with a reverse phase C 18 column using a gradient of 0-60% CH 3 CN in 0.1% TFAZH 2 O over 30 minutes and monitored at 220 nm.
  • TFA trifluoroacetic acid
  • Peptide labeling reactions were also carried out at various pHs ranging from 5.0 to 8.5 with various buffering reagents (pH 5.0, sodium acetate 50 mM; pH 6.0, MES, 50 mM; pH 7.0 HEPES 50 mM; pH-8.0, HEPES 50 mM; pH 8.5 Tris-HCl 50 mM) to test the effect of pH on the rate of Sfp catalyzed peptide labeling.
  • buffering reagents pH 5.0, sodium acetate 50 mM; pH 6.0, MES, 50 mM; pH 7.0 HEPES 50 mM; pH-8.0, HEPES 50 mM; pH 8.5 Tris-HCl 50 mM
  • the peptide concentration was kept constant at 500 ⁇ M and the biotin - CoA or fluorescein - CoA concentration was varied from 2 ⁇ M to 200 ⁇ M.
  • the reaction was
  • the kinetic parameters for the Sfp catalyzed PCP labeling was carried out in the same buffer (10 mM MgCl 2 and 50 mM HEPES, pH 7.5) in presence of 0.1 ⁇ M of Sfp by either varying the concentration of PCP from 0.5 ⁇ M to 50 ⁇ M at a constant biotin - CoA concentration of 150 ⁇ M or varying the biotin - CoA concentration from 2 ⁇ M to 200 ⁇ M at a constant PCP concentration of 20 ⁇ M.
  • the reaction was allowed to
  • reaction proceed at 37°C for 5 minutes before quenching by the addition of 30 ⁇ L 4% TFA to 100 ⁇ L reaction mixture.
  • the reaction was then analyzed by analytical HPLC with a reverse phase C 18 column using a gradient of 30-50% CH 3 CN in 0.1% TFAZH 2 O over 30 minutes and monitored at 280 nm.
  • Peptides e.g., alpha helical oligopeptide moieties
  • TFE 2,2,2-trifluoroethanol
  • All the peptides had a concentration of 0.2 mg/ml, except for the TycC3 peptide, and were prepared to a concentration of 0.1 mg/ml for solubility purposes.
  • Circular dichroism ellipticity was recorded using an AVIV 60DS instrument. Spectra from 260 nm to 190 nm were scanned at a step of 1 nm at 25 0 C in a 0.1 cm cuvette, with 3 repeats and an averaging time of 3 seconds. The data were finally expressed as the mean residue molar ellipticity, The helix contents of peptides were estimated by Greenfield and Fasman equation (Plenum Press, New York, 1996).
  • Jun233 (5'- TCC AGC CCC CAT ATG GGA GAT TCT CTT TCG TGG CTG CTT AGG TGT TTG AAT GG) (SEQ 3D NO:48) and Jun234 (5'- TCC AGC CCC CAT ATG GGA GAT TCT CTT TCG TGG CTG CTT AGG CTT TTG AAT GGT AAG C) (SEQ ID NO:49) were then used to pair with Juii57 in order to amplify 5'- S2 and S6 fused EGFP genes from vector pET22b-Sl-EGFP.
  • PCR fragments were cloned into pET22b vector to give plasmids ⁇ ET22b-S2-EGFP and pET22b-S6 ⁇ EGFP for the expression of N-terminal S2 and S6 tagged and C-terminal 6x His tagged EGFP proteins.
  • Jun221 (5'- TTG TTA GCA GCC GGA TCC TCA ATT CAA ACA CCT AAC CAG CCA CGA AAG AGA ATC TCC GCC CTC GAG CTT GTA CAG C) (SEQ ID NO:50) and Juii222 (5'- TTG TTA GCA GCC GGA TCC TCA CAT CAA AGA CCA CTC CAA CAT ATC AAG AGA ATC TCC GCC CTC GAG CTT GTA CAG C) (SEQ ID NO:51) were paired with Junl76 (5'- TAA TAC GAC TCA CTA TAG GG) (SEQ ID NO:52) in order to amplify 3'- Sl and Al fused EGFP genes from vector pET14b-EGFP-ybbR12 (Yin et al., Proc.
  • peptide-tagged EGFP protein for 15 minutes.
  • 20 ⁇ L labeling reaction mixture was added to 80 ⁇ L 1% BSA in TBS in the wells of a streptavidin coated 96 well plate.
  • Cross-plate series dilution of the labeling reaction mixtures were performed similar to phage ELISA and the plate was incubated at room temperature for 60 minutes and washed five times with 0.05% (v/v) Tween 20, 0.05% (v/v) Triton X-100 in TBS and five times with TBS.
  • Biotinylated EGFP bound to the streptavidin surface were detected by incubation with 1/200 diluted mouse anti-GFP antibody (SANTA CRUZ BIOTECHNOLOGY) followed by incubation with 1/5000 diluted goat anti-mouse IgG antibody HRP conjugate. The plate was then developed by ! adding 100 ⁇ L TMB peroxidase substrate and hydrogen peroxide mixture (PIERCE) to each well and incubated at room temperature for five minutes before photography. Biotin labeling of S and Al tagged EGFP in the cell lysates and streptavidin pull down of biotinylated EGFP was performed as previously reported . (Id. Yin et al.)
  • transfection medium was prepared for each 35-mni well in a 6-well plate. Each vial contained 3 ⁇ l of FuGENE 6 transfection reagent (ROCHE DIAGNOSTICS CORPORATION) and 2 ⁇ g of the relevant plasmid diluted in 100 ⁇ l of Dulbecco's modified Eagle medium/nutrient mixture F-12 (Ham) (DMEM/F12) (1:1). The transfection medium was mixed and allowed to come to equilibrium over an additional 20 minutes. TRVb cells were grown on sterilized coverslips to 50-60% confluency, incubated with transfection medium for 5 hours, and then incubated in standard cell media (with serum) for 24 hours to allow time for protein expression.
  • FuGENE 6 transfection reagent ROCHE DIAGNOSTICS CORPORATION
  • DMEM/F12 Dulbecco's modified Eagle medium/nutrient mixture F-12
  • TRVb cells transfected with TfRl-Al were incubated in serum-free media for two hours prior to labeling.
  • TfRl-Al cells were then incubated with 1.98 ⁇ M Acps and 1 ⁇ M CoA-Alexa Fluor 488 in serum-free media for 30 minutes at 37°C. Labeled cells were washed three times with PBS, then incubated with 10 ⁇ g/ml Alexa Fluor 568-conjugated Tf (MOLECULAR PROBES INC.) for 5 minutes and washed three times with PBS. Finally, cells were fixed using a 3.7% formaldehyde solution in
  • Example 1 vbbR is a substrate for Sfp
  • the truncated forms of the predicted ybbR protein corresponding to amino acids 95-278 (JY565), 111-278 (JY503), 214-278 (JY530) and 229-278 (JY529) were selected multiple times by Sfp catalyzed biotin labeling followed by streptavidin binding.
  • phages expressing various lengths of the displayed ybbR protein were labeled in the presence of both Sfp and biotin — CoA and added to a 96-well streptavidin plate in a phage enzyme linked immunosorbant assay (ELISA). After washing, the phages which bound to a streptavidin plate were detected by anti-M13 phage antibody conjugated with horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • JY529-His expressed as a fusion protein containing a C-terminal 6x Histidine tag (JY529-His).
  • Example 2 Serine274 in ybbR protein is the site of Ppant modification by
  • ybbR JY529-His The site of Ppant modification in ybbR JY529-His was identified by Fourier- Transform Mass Spectroscopy (FTMS). Truncated ybbR protein JY529-His was covalently modified by Ppant group in presence of CoA and Sfp.
  • the unmodified (apo) and Ppant modified (holo) forms of JY529-His were separated by high pressure liquid chromatography (HPLC) and introduced into the mass spectrometer, resulting in the detection of two species of about 8,513.58 Da and 8,853.37 Da in mass, which corresponded to the mass of the apo and holo forms of JY529-His, as depicted in Figure 4a within 0.36 Da and 0.06 Da (42 ppm and 7 ppm), respectively.
  • the 9 + charge state of holo - JY529-His was mass selected, as shown in Figure 4b, and fragmented using three MS/MS techniques.
  • Ser274 in JY529-His was subsequently mutated to Ala by site directed mutagenesis and the Ser274Ala mutant of JY529-His was shown as not being labeled with biotin after incubation with Sip and biotin - CoA as shown by Western blot analysis in Figure 3, confirming the FTMS assignment of Ser274 as the Ppant modification site in JY529-His.
  • JY529 which includes a ybbR tag and a C-terminal ⁇ xHistidine tag, did not show any significant sequence homology with any known ACPs or PCPs that are known substrates of Sfp.
  • the site of Ppant modification in JY529-His, i.e., Ser274 was very close to the C-terminus of the protein, in contrast to the Ppant modified Ser in ACPs or PCPs, which is in the middle of the 80-90 amino acid residue protein with 40-45 residues on either side of the Ser.
  • short peptides corresponding to the flanking sequence of Ser274 in JY529-His were synthesized, as depicted in Figure 5 a, and assayed for Sfp catalyzed Ppant modification.
  • the peptides were incubated with Sfp and biotin - CoA and the reaction mixture was assayed by HPLC.
  • Peptide ybbR13 (DSLEFIASKLA) (SEQ ID NO:2) was found to be modified by biotin — CoA in presence of Sfp, as shown by a product peak with a retention time of 21 minutes on HPLC trace 1, shown in Figure 6a.
  • the product formation was dependent on the presence of both biotin — CoA and Sfp since no product was formed when either biotin - CoA or Sfp were excluded from the labeling reaction (trace 2 and 3 in Figure 6 a).
  • Matrix - assisted laser desorption ionization mass spectroscopy confirmed that the product of Sfp catalyzed ybbRl 3 modification by biotin - CoA had the same mass as biotin - Ppant conjugated ybbRl 3 ([M+H] + , calculated 2058.0, observed 2058.2).
  • peptides of seventeen and thirteen residues in length - ybbRl 1 (GS QDVLD SLEFIASKLA) (SEQ E) NO:27) and ybbR12 (VLD- SLEFIASKLA) (SEQ ID NO:28) included nine residues LEFIASKLA (SEQ ID NO:29) which extended beyond the Ppant modified Ser274 (underlined), and were Biotin- labeled by Sfp, as shown by HPLC and MALDI (data not shown), hi contrast, peptides with fewer than seven amino acid residues beyond Ser274, e.g., ybbR3 (GSQDVLDSLEFI) (SEQ ID NO:30), ybbR8 (DVLDSLEFI) (SEQ ID NO:31)and ybbR14 (VLDSLEFIAS) (SEQ ID NO:32), were not Biotin-labeled in a Sfp catalyzed reaction, denoting the importance of the C
  • peptide sequence ASKLG was part of the linker between residue Ile278 and pill capsid protein.
  • Peptide ybbR15 VLDSLEFIDGVSL
  • SEQ ID NO:35 which had the original ybbR sequence flanking the Ppant modified Ser274 and the same number of residues beyond Ser274 as in Sfp active peptides ybbRl 1-13, failed to be the substrate of Sfp, suggesting the full length ybbR protein in B. subtilis proteome is not a substrate of Sfp.
  • Peptides ybbRll - 13 have the same nine amino acid residues (LEFIASKLA) (SEQ ID NO:29) C-terminus to Ppant modified Ser274 but have a different number of amino acid residues N-terminus to the Ppant modified Ser274, for example, seven amino acid residues in ybbRll, three amino acid residues in ybbR12 and only one amino acid residue in ybbR13. Notably, all three peptides showed
  • the ybbR peptides previously tested for Sfp catalyzed modification were dissolved in 5 niM potassium phosphate buffer, pH 7.5 with 30% 2,2,2-trifluoroethanol (TFE) and the peptide conformation was measured by circular dichroism (CD) spectroscopy.
  • CD circular dichroism
  • ybbRl 4 and ybbRl 5 exhibited a lesser extent of ⁇ -helical structure and more undefined
  • Example 5 ybbR peptides can be used for site-specific labeling of proteins [00169]In order to determine whether Sfp active ybbR peptides could be used as tags for site specific protein labeling, 13 residue ybbR12 peptide (VLDSLEFIASKLA) (SEQ ID NO:37) was fused either to the N- terminus or the C-terminus of enhanced green fluorescent protein (EGFP), glutathione-S-transferase (GST) and maltose binding protein (MBP), and the purified fusion proteins were labeled with biotin-Ppant in the presence of Sfp and biotin — CoA.
  • VLDSLEFIASKLA 13 residue ybbR12 peptide
  • EGFP enhanced green fluorescent protein
  • GST glutathione-S-transferase
  • MBP maltose binding protein
  • Biotin labeling of all ybbR tagged fusions in the cell lysates was confirmed by Western blot analysis and the control reactions where either biotin - CoA or Sfp was excluded did not give any biotin labeling, as shown in Figure 9, nor did the control reactions in which target protein without the ybbR tag was incubated with biotin - CoA and Sfp (data not shown), suggesting that the labeling reaction is strictly dependent on Sfp catalyzed biotin Ppant transfer onto Ser274 in the ybbR peptide.
  • PCP and ACP domains have a conserved (H/D)S(L/I) motif at the site of Ppant modified Ser (underlined) with the residue preceding the conserved Ser as a His or Asp and the residue following the Ser a Leu or He (Marahiel et al., Chem. Rev., 97:2651-2674 (1997)). Therefore, for the peptide library to be displayed on the phage surface, the DS sequence in the original ybbR peptide was retained. A Leu or He amino acid residue was maintained at the site immediately following the Ppant modified Ser by using a combination of CTT and ATT codons in the library.
  • the final size of the library was approximately 1 x 10 9 .
  • the peptide library was selected in parallel in separate tubes by Sfp- and Acps- catalyzed biotin-Ppant attachment to the phage-displayed peptides using biotin-SS-CoA as the substrate, as shown in Figures 12b and 12c. After the first round of selection, the library diverged in the subsequent rounds, in that phages selected by Sfp were exclusively used for the next round of Sfp selection and phages selected by Acps were exclusively used for the next round of Acps selection.
  • phage particles were precipitated by polyethylene glycol (PEG) to quench the reaction and remove unreacted biotin-SS-CoA followed by resuspension in 1% bovine serum albumin (BSA) in Tris buffered saline (TBS), pH 7.4.
  • PEG polyethylene glycol
  • BSA bovine serum albumin
  • TBS Tris buffered saline
  • the phage particles displaying peptides as the substrates of Sfp or Acps were covalently conjugated to biotin through the Ppant arm linked to a disulfide bridge and subsequently the phages were allowed to bind to streptavidin-coated 96 well plates, as shown in Figure 12c.
  • biotin- conjugated phage particles were cleaved from the solid support by incubating with 20 mM dithiothreitol (DTT) in TBS. The eluted phage particles were then rescued by infecting E. coli XLl -Blue cells and carried on for the next round of selection.
  • DTT dithiothreitol
  • controls were performed excluding the enzymes or biotin-SS-CoA in the labeling reaction before binding the phage particles to the streptavidin plates. There was a steady increase in the ratio of phage recovery for the reaction with the addition of both the enzyme and biotin-SS-CoA over the controls, as depicted in Figure 13.
  • the ratio of the reaction over the controls was more than 10 4 , suggesting the selection of phage particles by streptavidin binding was indeed dependent on the enzyme Sfp or Acps to catalyze biotin labeling of the phage displayed peptides and that the peptide clones enriched after the fifth round of selection could be very efficient substrates of Sfp and Acps.
  • peptide clone Sl and a closely related sequence Sl' with two mutations appeared a total of 6 times among the 30 sequencing samples from the Sfp-selected libraries; similarly, peptide clone Al was counted 8 times among the 40 sequencing samples from the Acps-selected library. This suggested that Sl and Al clones started to dominate the final selected pool of peptides after the fifth round of selection using Sfp and Acps, respectively, and thus the phage selection was stopped at the fifth round.
  • Asps selected phage clones Name Peptide sequence Number of occurrences
  • Example 9 Kinetic characterization and mutagenesis studies of the selected peptides.
  • peptides Sl, S4, S5, S9 identified from the Sfp selection of the phage-displayed peptide libraries and peptides Al , A2, A3 and A4 from the Acps selection were synthesized as 12-mers, all starting with the GDSL sequence flanking the Ppant modified Ser (underlined). Sequence alignment data revealed that one of the S series of peptides, e.g., Sl', selected by Sfp selection, differed from the Sl peptide at position 8 as it had a Leu instead of a VaI amino acid residue (Ppant modified Ser is designated position 3).
  • Peptide S2 was synthesized as having a VaI to Leu mutation at position 8. It was speculated that because peptide Sl has a Cys residue at position 10, that the Cys residue may introduce complexities for disulfide formation, should Sl be used as a tag for protein labeling. Accordingly, peptide S3 was synthesized with a Cys to Leu mutation at position 10 in the Sl peptide and S6 was synthesized with the same mutation in the S2 peptide, as depicted in Table 3. The amino acid residue Cys was replaced by Leu because some peptides selected by Sfp had a Leu residue at the same position, as shown in Figurel4.
  • the Al peptide showed activity for Acps-catalyzed peptide labeling, with a k cat /K m of 0.015 ⁇ M ⁇ min "1 and a K m of 117 ⁇ M, comparable to the activities of the S peptides for Sfp-catalyzed protein labeling.
  • Corresponding peptides from the phage clones enriched by Sfp selection, S4, S5 and S9, and from Acps selection, A2, A3, and A4 were also synthesized. Their specific activities with Sfp and Acps respectively were more than 5- to 10-fold lower than the S6 and Al peptides, consequently, detailed kinetic characterizations were not carried out with them.
  • the S6 peptide showed the highest level of differentiation for different PPTases, with a 442-fold higher k cat /K m for Sfp-catalyzed peptide labeling than the Acps-catalyzed reaction, hi contrast, the ybbR13 peptide, identified as a Sfp substrate, showed substantial activity with Acps, with a k ca t/K m of 0.0033 uM ' Wn "1 , only 28-fold lower than the Sfp-catalyzed reaction.
  • S6 appears to be a better tag for Sfp labeling than ybbR not only because it has a lower K m and higher activity as the substrate of Sfp but also because it shows more than 7-fold lower activity (k cat /K m ) than ybbR for Acps- catalyzed modification.
  • Both the S6 and ybbR peptides show a preference for Sfp over Acps; however, the S6 peptide showed a greater than about a 440-fold preference as a substrate of Sfp versus Acps, while the ybbR tag showed a preference for Sfp ⁇ f only 28-fold over Acps.
  • A1 was a poor substrate for Sfp, with a WK n , of 0.00049 ⁇ M ' Wn "1 , 30-fold lower than that of the reaction catalyzed by Acps.
  • S6 and Al two additional peptides were identified, S6 and Al, which are good substrates for the peptide labeling reactions catalyzed by Sfp and Acps, respectively.
  • S6 and Al also show significant orthogonality in enzyme differentiation, with S6 a preferred substrate of Sfp and Al a preferred substrate of Acps.
  • S6 or ybbR and Al could be used for simultaneous labeling of molecules, by simultaneous Sfp and Acps catalyzed reactions, respectively.
  • Table 3 Kinetic characterization of S ⁇ - and AcpS-catalyzed peptide labeling reaction by biotin-CoA (2). Ppant modified Ser is underlined. Mutations made to the Sl peptide sequence were shown in bold.
  • Example 10 Use of S6 and Al Peptides for Protein Labeling.
  • S6 and Al 12-mer peptide were evaluated as peptide tags fused to target proteins.
  • the Sl and Al peptides were fused to either the N or C termini of the enhanced green fluorescence protein (EGFP) and tested for biotin labeling with EGFP as the target protein. Later, the optimized S2 and S 6 tags were also fused to the N-terminal of EGFP.
  • EGFP enhanced green fluorescence protein
  • Sl-, S2-, S6- and Al-tagged EGFP were tested for biotin labeling by both S ⁇ and Acps using biotin-CoA as the substrate, and the labeling results were compared to biotin labeling of ybbR- or PCP- tagged EGFP.
  • Control reactions were also run with the exclusion of enzymes or both enzymes and biotin-CoA.
  • the labeling reaction mixture was loaded on the 96-well streptavidin plate and a 5 -fold series dilution was carried out across the wells in the plate. After washing, biotinylated GFP immobilized onto the streptavidin surface was detected using a mouse anti-GFP antibody and a goat anti-mouse antibody-HRP conjugate.
  • N-terminal Sl-, S2- or S ⁇ -tagged EGFP proteins were efficiently labeled with biotin by Sfp using biotin-CoA as the substrate.
  • N-terminal Al -tagged EGFP was efficiently labeled with biotin by Acps.
  • the ELISA signals for Acps- catalyzed biotin labeling of EGFP fused to Sl, S2 or S6 tags were more than 100-fold lower than the same labeling reactions catalyzed by Sfp, suggesting that the S-tagged proteins were not labeled by Acps efficiently.
  • Al -tagged EGFP was not labeled with biotin by Sfp very efficiently, as shown by ELISA.
  • S tags and the Al tag can be used as orthogonal tags for target- specific protein labeling catalyzed by Sfp and Acps, respectively.
  • the S and Al peptide tags were also fused to the C-terminal of EGFP or to the N-terminal of glutathione S- transferase (GST) and maltose binding protein (MBP). All showed similar labeling efficiency, with S-tagged proteins preferentially labeled by Sfp and Al -tagged proteins preferentially labeled by Acps (data not shown), denoting the portability of the S and Al tags for the construction of fusions at N- or C-termini of various target proteins.
  • Figure 18 also shows the biotin labeling of N-terminal ybbR- or PCP-tagged EGFP catalyzed by Sfp or Acps.
  • PCP-tagged EGFP showed preferential labeling by Sfp, although ybbR-tagged EGFP showed significant labeling by both Sfp and Acps, suggesting that the S tags are more specific as substrates for Sfp-catalyzed protein modification than the ybbR tag.
  • S and Al tagged EGFP can also be labeled with biotin in the cell lysates by Sfp and Acps, respectively, and the biotinylated protein can be detected by ELISA (data not shown).
  • S - or Al -tagged EGFP proteins were labeled with biotin by Sfp and Acps followed by the addition of streptavidin coated agarose beads to pull down the biotin-labeled EGFP. More than 80% of the S- or Al -tagged EGFP could be immobilized on the streptavidin beads after the biotin labeling reaction catalyzed by S ⁇ or Acps, respectively. On the other hand, less than 5% of the EGFP could be pulled down by streptavidin beads in the cross- labeling reaction using S ⁇ to label Al -tagged protein or Acps to label S-tagged protein.
  • Tf-TfRl transferrin-transferrin receptor 1
  • Al peptide tag The transferrin-transferrin receptor 1 (Tf-TfRl) system was used to demonstrate surface protein labeling of cells using the Al peptide tag.
  • TfR-mediated iron uptake represents the major mechanism used by vertebrate cells to acquire iron from the environment, and the endocytic cycle of Tf-TfRl has been well established (Klausner et al, Proc. Natl. Acad. Sci. USA, 80:2263-2266 (1983); Morgan, MoI. Aspects Med., 4: 1-123 (1981)).
  • TfRl-Al was transiently transfected into TRVb cells, a Chinese hamster ovary cell line that lacks endogenous TfRl, and labeled with CoA-conjugated Alexa Fluor 488 in the presence of Acps. Transfected cells were then incubated with Alexa Fluor 568 diferric human transferrin conjugates (Tf- Alexa 568) for 5 minutes, and fixed for observation under the confocal microscope. Imaging of transferrin receptor 1 using the Al peptide tag was performed.
  • TRVb cells were transfected with TfRl-Al and then incubated with AcpS and Co A- Alexa Fluor 488 for 30 minutes, washed, and incubated with Alexa Fluor 568-labeled transferrin for 5 minutes. Cells were fixed and imaged using confocal microscopy. Stacks of optical slices at 0.25 ⁇ m per step were processed by MetaMorph using 2D no-neighbors deconvolution followed by 3D reconstruction. Colocalization of TfRl-Al and Tf was observed (data not shown). [00191 ] Accordingly, these results demonstrate that the Al peptide tag may be used for surface protein labeling in mammalian cells in order to monitor the interactions and trafficking of cell surface receptors while allowing these receptors to maintain their cellular function.
  • Example 12 Comparison of the S, A and vbbR peptides from the phage selection for Sfp and Acps Modification.
  • the ybbR13 peptide has a Phe residue at position 6 and correspondingly phage selection of the peptide library in this work leads to an aromatic Trp residue at the same position in S6.
  • the ybhR13 peptide has a basic Lys residue at position 10 while the current, independent selection leads to an Arg side chain at position 9 in close proximity in S6 peptide.
  • S6 and ybbR13 share very similar organization for the assembly of ⁇ -helix and since the hydrophobic side of the helix is preserved in the selection. It is contemplated that the helix mimics the neutral protein surface of PCP upon the interaction of peptides S6 and ybbR13 with Sfb.
  • S6 was derived from peptide Sl, which was the most abundant clone enriched by phage selection using Sfp. Two mutations in Sl, VaI to Leu at position 8 and Cys to Leu at position 10, gives S6 which is kinetically a more active substrate for Sfp. These mutations are both on the hydrophobic side of the helix and give a condensed cluster of Leu side chains on this side of the ⁇ -helix.
  • S6 has a twofold lowering of K m compared to ybbR13 and Sl peptides and a higher tendency to adopt an ⁇ -helical conformation than Sl, suggesting the formation of the hydrophobic face of the ⁇ -helix may well contribute to the binding of S6 peptide to Sfp and the subsequent peptide modification reaction.
  • Al peptide does not have significant ⁇ -helical conformation in 30% TFE according to its CD spectrum ( Figure 19). Without wishing to be bound by theory, it is, nevertheless contemplated that the Al peptide forms an ⁇ -helical conformation upon binding to Acps.
  • Al shows a very different helical surface from those of the Sl, S6 and ybbR13 peptides ( Figure 20).
  • Al also has Leu side chains at positions 4, 7, 11- which may promote helix formation
  • Al has negatively charged Asp and GIu side chains at positions 5 and 8, respectively, and the polar residue Ser at positions 3 and 10, which makes the hydrophobic side of Al considerably smaller. It is contemplated that this property may make the Al peptide a poor substrate of Sfp.
  • Acps is a highly positively charged protein with a pi value of 9.6 (Id.

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Abstract

The present invention relates to methods of site-specific labeling of molecules and molecules produced thereby. The methods described herein are based, at least in part, on the discovery of novel peptide substrates for phosphopantetheinyl transferases, which can be used for site-specific labeling of both proteinaceous and non-proteinaceous molecules using a phosphopantetheinylation reaction.

Description

METHODS OF SITE-SPECIFIC LABELING OF MOLECULES AND MOLECULES PRODUCED THEREBY
Government Support
[0001 ] Work described herein was supported in part by NIH grants HL32854, HL70819 and GM20011. The United States government therefore may have certain rights in the subject matter disclosed herein.
Related Applications
[0002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 60/723,640, filed October 4, 2005, the entire contents of which are incorporated by reference herein.
Background of the Invention
[0003] Incorporation of site-specific probes into biological molecules such as proteins is particularly desirable for investigating such molecules in their natural habitat and for studying conformational changes in structure and expression and translocation of such molecules in cells.
[0004] A few methods for site-specific labeling of proteins have been described. For example, O6-alkylguanine-DNA alkyltransferase (AGT) can be fused to a target protein of interest, followed by the addition of a fluorescently-labeled O6-benzylguanine suicide substrate for the AGT. (Keppler, A. et al. Nat. Biotechnol. 21, 86-89, 2003). However, the AGT tag is 207 amino acids long and introduces a large amount of steric bulk. While, smaller peptide tags are generally more desirable, they typically lack specificity. For example, cysteine labeling is not at all specific inside cells, and tetracysteine labeling (Griffin, B A et al, Science 281, 269-272, 1998), while better, is still insufficiently specific for most applications and allows only a small set of probes to be introduced. Transglutaminase has also been used to label glutamine side chains in proteins with fluorophores in vitro (Sato, H. et ah, Biochemistry 35, 13072-13080, 1996), however it is relatively promiscuous for peptide and protein substrates, precluding its use in mammalian cells.
[0005] Recently, intein-based methods were described for attaching a wide array of small molecules to proteins including, for example, fluorophores, carbohydrates, oligonucleotides, affinity tags and metal chelators. (Muir, T. et ah, Proc. Natl. Acad. Sci. U.S.A., 95, 6705-6710, 1998). However, a major drawback of the intein-based methods is the size of the intern domain itself, which is about 454 amino acids long. Other disadvantages of the intein-based methods include, for example, the length of the reaction which typically requires an overnight incubation. (Id.) [0006] Phosphopantetheinyl transferase based reactions have also been utilized for labeling fusion proteins containing a PCP excised from a nonribosomal peptide synthetase. (Yin et ah, J. Am. Chem. Soc, 126:7754-7755 ,2004). A major drawback of using PCP for site specific labeling of proteins, however, again is the size of PCP, i.e., about 80-100 amino acids.
[0007] Therefore, there is a need for developing methodologies for site-specific labeling of biological molecules such as proteins, which avoid the various aforementioned drawbacks of the currently available methodologies.
Summary of the Invention
[0008] The present invention relates, at least in part, to the discovery of novel peptide substrates for phosphopantetheinyl transferases, such as, for example, Sfp and Acps, which can be used for site-specific labeling of both proteinaceous and non-proteinaceous molecules. In some embodiments, the present invention provides methods for site- specific labeling of polypeptides, and labeled polypeptides produced by such methods. Also, provided are kits including phosphopantetheinyl transferases and substrates for labeling of molecules, and methods of using labeled molecules produced by the methods described herein.
[0009] Li some embodiments, the present invention relates to a method of generating a molecule of formula (I):
Mq-L-Y-Z (I) wherein:
M is a first agent; q is an integer greater than 0;
L is a linking group;
Y is an alpha-helical oligopeptide moiety; and Z is a second agent; comprising: a) providing a compound of formula (II):
Y-Z (II) wherein:
Y is the alpha-helical oligopeptide moiety; Z is the second agent; and b) contacting the compound of formula (II) with an enzyme having phosphopantetheinyl transferase activity or a fragment thereof having phosphopantetheinyl transferase activity, in the presence of a compound of formula (III):
Mq-L-N '(III) wherein:
M is the first agent; q is an integer greater than 0;
L is the linking group; and N is a leaving group; under suitable conditions to allow the enzyme or the fragment thereof to attach Mq-L to the alpha-helical oligopeptide moiety (Y), thereby generating the compound of formula (I).
[0010] In some embodiments, the present invention provides a method of generating a molecule of formula (VIII):
G-Y-Z (VIII) wherein:
G is a transferred moiety;
Y is an alpha-helical oligopeptide moiety; and Z is a second agent, comprising:
(a) providing a compound of formula (IX):
Y-Z (IX) wherein:
Y is the alpha-helical oligopeptide moiety; Z is the second agent; and
(b) contacting the compound of formula (IX) with an enzyme having phosphopantetheinyl transferase activity or a fragment thereof having phosphopantetheinyl transferase activity, in the presence of a compound of formula (X):
G-N (X) wherein:
G is the transferred moiety; and N is a leaving group; under suitable conditions to allow the enzyme or the fragment thereof to attach the transferred moiety (G) in formula (X) to the alpha-helical oligopeptide moiety (Y) in formula (IX), thereby to generate the molecule of formula (VIII). The transferred moiety generally includes a first agent linked to a linking group. [0011] Alpha-helical oligopeptide moieties that can be used in the methods described herein include those set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:40, and SEQ ID NO:41, and variants thereof, which are capable of being phosphopantetheinylated by an enzyme having phosphopantetheinyl transferase activity, e.g., Sfp and Acps. [0012] In some embodiments, the first agent used in the methods described herein is a chosen from the group consisting of small molecules moieties (e.g., drug moieties, toxins), protons, haptens, affinity probes, spectroscopic probes, radioactive probes, peptides, non-naturally occurring amino acids, nucleic acids, lipid molecules, radical generating molecules, singlet oxygen generating molecules, polymers, sugars (e.g., monosaccharides, disaccharides, polysaccharides, and other carbohydrate containing moieties), antibodies and antibody fragments, enzymes, enzyme substrates, chelating agents and receptor binding molecules.
[0013] The second agent used in the methods described herein can either be proteinaceous or non-proteinaceous. Examples of proteinaceous second agents include, for example, polypeptides, enzymes, fusion proteins, antibodies and hormones. Examples of non-proteinaceous second agents include, but are not limited to, a nucleic acid molecule and derivatives thereof, a carbohydrate and derivatives thereof, and a lipid molecule and derivatives thereof. A second agent may also be a small molecule moiety. [0014] In some embodiments, the present invention provides a method of generating a protein-small molecule conjugate of formula (XI):
Iq-L-Y-K (XI) wherein:
I is a small molecule; q is an integer greater than 0;
L is a linking group;
Y is an alpha-helical oligopeptide moiety; and K is a protein; comprising: a) providing a compound of formula (XII):
Y-K (XII) where:
Y is the alpha-helical oligopeptide moiety; and K is the protein; and b) contacting the compound of formula (XII) with an enzyme having phosphopantetheinyl transferase activity or a fragment thereof having phosphopantetheinyl transferase activity, in the presence of a compound of formula (XIII):
Iq-L-N (XIII) where:
I is the small molecule; q is an integer greater than 0; L is the linking group; and N is a leaving group; under suitable conditions to allow the enzyme or the fragment thereof to attach the small molecule (Iq) to the alpha-helical oligopeptide moiety (Y) via the linking group (L) thereby generating the protein-small molecule conjugate of formula (XI). [0015] Also encompassed by the present invention are molecules produced by the methods described herein. For example, in some embodiments, the present invention provides a molecule of formula (IV):
Mq-L-Y-Z (IV) where:
M is a first agent; q is an integer greater than 0;
L is a linking group;
Y is an alpha-helical oligopeptide moiety; and
Z is a second agent. [0016] Also encompassed by the present invention are pharmaceutical compositions containing molecules produced by the methods described herein, hi some embodiments, i pharmaceutical compositions described herein may further include a pharmaceutically acceptable carrier.
[0017] Further encompassed by this invention are methods of using the molecules produced by the methods described herein. For example, in some embodiments, a method of delivering a second agent such as, for example, a protein, to a desired location in a subject is described. A method of delivering a second agent to a desired location within a subject, includes for example, administering to a subject a molecule of formula (T), where the first agent is capable of delivering the second agent to the desired location within the subject, where the molecule of formula (T) is:
Mq-L-Y-Z (I) where:
M is a first agent; q is an integer greater than 0; L is a linking group;
Y is an alpha-helical oligopeptide moiety; and
Z is a second agent.
[0018] In some embodiments of the present invention, a method of labeling two target proteins is provided. Such a method includes (a) providing a first protein and a second protein, where the first protein is linked to at least one alpha helical oligopeptide moiety including an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40, and the second protein is linked to at least one alpha helical oligopeptide moiety including an amino acid sequence set forth in SEQ ID NO:41; and (b) contacting the first protein and the second protein with one or more compounds in the presence of one or more phosphopantetheinyl transferase enzymes chosen from Sfp, Acps, or a fragment thereof having phosphopantetheinyl transferase activity, under conditions such that to allow the enzyme to transfer the one or more compounds to the one or more alpha-helical oligopeptide moieties in step (a).
[0019] In a method of labeling a first and a second protein, the Sfp enzyme or a fragment thereof having phosphopantetheinyl transferase activity transfers a compound to an alpha-helical oligopeptide moiety having an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40, thereby to label the first protein and the Acps enzyme or a fragment thereof having phosphopantetheinyl transferase activity transfers a compound to an alpha-helical oligopeptide moiety having an amino acid sequence set forth in SEQ ID NO:41, thereby to label the second protein.
[0020] In one or more embodiments, the compound includes the formula Iq-L-N (XIII), where:
I is the small molecule; q is an integer greater than 0; L is the linking group; and
N is a leaving group;
[0021 ] The first and the second protein may either be labeled simultaneously or sequentially. Also, the first and the second protein may either be labeled with the same compound or each of the first and second protein may be labeled with different compound.
[0022] The first and the second protein may either be present in a cell lysate or be expressed on the surface of live cells.
Brief Description of the Drawings
[0023] Figure 1 depicts Sfp catalyzed PCP or ybbR tag modification at a specific Serine residue by various small molecule-CoA conjugates. Rl depicts biotin-CoA conjugate; R2 depicts fluorescein-CoA conjugate; R3 depicts tertramethylrhodamine- CoA conjugate and R4 depicts Texas Red-CoA conjugate. [0024] Figure 2 depicts amino acid sequence alignment of B. subtilis ybbR open reading frame (amino acids 1-299) with the truncated ybbR clones JY503, JY529, JY530 and JY565, selected by phage display. Sequences that match are highlighted. Truncated ybbR amino acid sequences are preceded by a leader peptide followed by the sequence of the phage capsid protein pUI. The Ppant modified Serine residue at position 274 is boxed.
[0025] Figure 3 depicts results of a Western blot analysis of JY529-His labeled with biotin by Sfp catalyzed biotin-CoA modification. A Ser274Ala mutant of JY529-His was used as a control and the Western blot was probed with streptavidin-HRP. [0026] Figure 4 depicts localization of the Ppant modified serine in ybbR JY529-His using Q-FTMS and tandem mass spectrometry. Figure 4a is the charge state distribution for intact Ppant modified JY529-His (8,853.37 Da). Figure 4b depicts mass selection of the 9+ charge state of Ppant modified JY529-His. Figure 4c shows fragment ion profiles for JY529-His. Figure 4d depicts the y23 ion, generated using IRMPD. This ion is 80 Da greater in mass, consistent with phosphate retention. Figure 4e shows the y20 ion generated using IRMPD. Similar to the previous ion, this species is 80 Da greater in mass. Figure 4f shows the yl7 ion, generated using IRMPD. This ion possesses its normal mass. The three ions in 4d, 4e and 4f indicate that the highlighted and underlined Ser274 is the site of covalent modification.
[0027] Figure 5a shows alignment of ybbR peptides with peptide sequences flanking the Ppant modified Ser (in box) in known PCPs and ACPs. The conserved Asp and Leu residues at the site of Ppant modification are highlighted. The position of helix II based on the NMR structures of TycC3-PCP and FrenN-ACP is also shown. Figure 5b shows helical wheel plots of ybbR peptide and the helix II region of TycC3-PCP and FrenN- ACP starting with the Ppant modified Ser residue (underlined). Solvent exposed side of helix II in TycC3-PCP and FrenN-ACP are shaded.
[0028] Figure 6 depicts the kinetic analysis of Sfp catalyzed peptide ybbR13 modification by biotin CoA. Figure 6a depicts the HPLC traces of the ybbR13 peptide labeling reaction with biotin CoA and Sfp added (trace 1) and the control reactions with either Sfp (trace 2) or biotin-CoA (trace 3) excluded from the reaction mixture. Figure 6b depicts a Michaelis - Menten plot for the measurement of kinetic parameters of Sfp catalyzed ybbR13 labeling at saturating concentration of biotin- CoA (150 μM). [0029] Figure 7 is a schematic of the NMR structures of TycC3-PCP (PDB ID IDNY) and FrenN-ACP (PDB ID 1OR5). Ser45 in TycC-PCP and Ser39 in FrenN-ACP at the tip of helix II were posttranslationally modified by Sfp.
[0030] Figure 8 depicts the results of a CD spectra of the various peptides in 5 mM potassium phosphate, pH7.5 with 30% TFE. Figure 8a summarizes the CD spectra analysis of the peptides ybbRl 1, ybbR12 and ybbR13 that were the substrates of Sfp. Figure 8b summarizes the CD spectra analysis of the peptides ybbR3, ybbR8, ybbRl 4 and ybbRl 5, that were not the substrates of Sfp. Figure 8c depicts the CD spectra analysis of peptides PksLl and TycC3 which include sequences that flank the Ppant modified Ser residue in PksL-PCP and TycC-PCP, respectively. [0031] Figure 9 depicts the results of a Western blot analysis of biotin labeled ybbR fusions A-L as shown in the table above. Labeling reactions were carried out in cell lysates in which (1) both biotin - CoA and Sfp were added; (2) only biotin-CoA added; or (3) only Sfp was added.
[0032] Figure 10a is a schematic of an EGFP-ybbR145 construct including a ybbR peptide in the middle. The Ppant modified Ser residue in the ybbR peptide is underlined. Figure 10b depicts a UV spectra of EGFP-ybbR145 labeled with tetramethylrhodamine or Texas Red by Sfp catalyzed ybbR labeling.
[0033] Figure 11 is a bar graph depicting the yield of the various labeled EGFP proteins including a ybbR peptide at the N-terminus, the C-terminus or in the middle. Yields of the protein labeling reactions were quantified by the binding of biotin labeled EGFP proteins to streptavidin beads. EGFP proteins with an N-terminal ybbRl 2 tag (N- ybbR12-EGFP), an N-terminal ybbR13 tag (N-ybbR13-EGFP), an N-terminal six residue peptide DSLEFI (N-DSLEFI-EGFP), a C-terminal ybbRl 3 tag (C-ybbR13- EGFP) and an internal ybbRl 2 peptide at residue 145 (I-ybbR12-EGFP) were labeled with biotin by Sfp and biotin-CoA and immobilized on streptavidin beads. The yields of the labeling reaction in terms of percentages of EGFP binding to streptavidin beads were compared to that of the N-terminal PCP-EGFP fusion (N-PCP-EGFP) and the control reactions with no Sfp added. [0034] Figure 12 is a schematic representation of the peptide labeling reaction and the selection scheme of the phage-displayed peptide libraries. Figure 12a is a schematic of an Sfp or Acps-catalyzed reactions resulting in PCP, ACP or peptide tag labeling using small molecule-coA conjugates as the donor of the small molecule-Ppant group to a specific Serine residue in PCP, ACP or the peptide tags. Figure 12b is a schematic depiction of structures of biotin-SS-CoA and biotin-CoA used in the selection and phage
ELISA. Figure 12c is a schematic representation of the selection of the peptide substrates of Sfp and Acps from phage displayed peptide library.
[0035] Figure 13 is a bar graph depicting steady enrichment of the phage clones for the selection of the phage displayed peptide library by Sfp over five rounds of selection.
Selection of the peptide library with AcpS showed similar results.
[0036] Figure 14 depicts an alignment of peptide sequences in the phage clones enriched by (a) Sfp- or (b) AcpS-catalyzed biotin labeling after the fifth round of selection. The GDS sequence was not varied in the peptide library.
[0037] Figure 15 depicts the consensus sequences of the peptides displayed by the phage clones enriched by Sfp (a) and AcpS (b) catalyzed biotin labeling after five rounds of selection. Ppant modified Ser is at position 3.
[0038] Figure 16 depicts the results of a phage ELISA from biotin labeling reactions with the phages displaying Sl and Al peptides.
[0039] Figure 17 depicts the results of a phage ELISA of the biotin labeling reactions with the phage clones enriched after the fifth round of selection. Phage clones S4, S5 and S9 were from the Sfp selection and A2, A3 and A4 were from the Asps selection.
20 μL of the phage labeling reaction was added to 80 μL of 1% BSA in TBS buffer in the first column of the streptavidin plate and cross plate 5 fold dilution was performed followed by phage detection using an anti-Mi 3 antibody-HRP conjugate. [0040] Figure 18 depicts the results of an ELISA of N-terminal A- and S peptide- tagged EGFP labeled with biotin by Sfp- or Asps-catalyzed protein modification. N- terminal ybbR- or PCP-tagged EGFP were used as the controls.
[0041] Figure 19 depicts a CD spectra of the A and S peptides.
[0042] Figure 20 is a schematic representation of the helical wheel conformation of the ybbR13, Sl, S6 and Al peptide tags. The Ppant modified Ser is underlined.
Detailed Description of the Invention
[0043] This invention is based, at least in part, on the identification of novel peptide substrates for enzymes having phosphopantetheinyl transferase activity such as for example, Sfp and Acps, and use of such substrates for site-specific labeling of both proteinaceous and non-proteinaceous molecules.
[0044] Sfp and Acps represent two classes of phosphopantetheinyl transferases (PPTases) that show differences both in their substrate specificity for the carrier protein domains and in their structures (Flugal et al, J. Biol. Chem., 275:959-968 (2000); Lambalot et al, Chem. Biol., 3:923-936 (1996)). The Sfp class of PPTases are about 230 residues in size and the crystal structure of Sfp suggests it has a twofold symmetry with the N- and the C-terminal halves of the molecule adopting similar folds, with the active site of the enzyme at the interface (Hodneland et. al, Proc. Natl. Acad. Sci. USA, 99:5048-5052 (2002); Koglin et al, Science, 312:273-276 (2006)). In contrast, Acps is about 120 residues in length, about half the size of Sfp, and the crystal structures of Acps show that the enzyme assembles into trimers and the ACP and CoASH binding sites are formed at the interface between each monomer (Reuter et al, Embo. J., 18:6823-6831 (1999); Chirgadze et al, Embo. J., 19:5281-5287 (2000)). It has been reported that Sfp exhibits a much broader substrate specificity than Acps in that Sfp can modify both PCP and ACP domains from nonribosomal peptides synthetases, polyketide synthases, and fatty acid synthases, while Acps modifies only ACP (Flugel et al, J. Biol. Chem., 275:959-968 (2000); Parris et al, Structure, 8:883-895 (2000); Mofid et al, J. Biol. Chem., 277:17023-17031 (2002)).
[0045] ACP and PCP carrier protein substrates of both kinds of PPTases adopt similar folds as four-helix bundle proteins with the serine residue to be modified by the Ppant prosthetic group at the top of the second α-helix, which has been shown to play an important role for interacting with Sfp and Acps (Hodneland et al, Proc. Natl. Acad. Sci. USA, 99:5048-5052 (2002); Chirgadze et al., Embo. J., 19:5281-5287 (2000); Quadri et al, Biochem., 37:1585-1595 (1998); Li et al, Biochem., 42:4648-4657 (2003)). Although there is not an obvious consensus sequence difference between PCPs and Acps, the most significant difference between the two is the electrostatic surface potential of the carrier proteins, with a neutral protein surface for PCPs and a negatively charged acidic surface for ACP domains in FAS and PKS systems (Parris et al, Structure, 8:883-895 (2000)).
[0046] The present invention is based, at least in part, on the isolation and characterization of peptides, referred to as ybbR13, and the S and A series of peptides, specifically, S6 and Al, which are efficient substrates of phosphopantetheinyl transferases such as, for example, Sfp and Acps, respectively. [0047] ybbR13 is an 11 amino acid residue peptide, which is a substrate of phosphopantetheinyl transferases such as, for example, Sφ. The ybbR13 peptide has an amino acid sequence of DSJLEFIASKLA, set forth in SEQ ID NO:2, and was isolated from a phage displayed library of the B. subtilis genome. A part of the sequence of the ybbR13 peptide is derived from a B. subtilis ORF, called ybbR, and it includes the DSL tripeptide sequence at the N-terminus, as shown in Figure 5 a, which is conserved in known substrates of Sfp, for example, PCP; but the ybbR peptide does not include the amino acid sequence, DxFFxxLGG (SEQ E) NO:3) at its N-terminus, which is found to be conserved in PCPs. Also described herein are modifications, truncations and variants of the ybbR13 peptide which can be used as substrates in phosphopantetheinylation reactions for site specific labeling of both proteinaceous and non-proteinaceous molecules.
[0048] Additional peptides described herein as efficient substrates for enzymes having phosphopantetheinyl transferase activity are the S series of peptides and the A series of peptides, designated as "S" or "A" based on their reactivity with Sfp or Acps, respectively. Exemplary S series of peptides include, but are not limited to, S6, which is an efficient substrate for Sfp, and exemplary A series of peptides include, but are not limited to, Al, which is an efficient substrate for Acps. Both S6 and Al peptides are 12 amino acid residues in length.
[0049] Because of the differences in specificities of ybbR and S6, which are efficient substrates for Sfp and Al, which is an efficient substrate for Acps, a pairing of ybbR or S6 with Sfp and Al with Acps, can be used for site-specific labeling of two separate proteins, for example, either in cell lysates and/or on live cell surfaces. Such site- specific labeling of two separate proteins maybe carried out either sequentially or simultaneously.
[0050] As described in the Examples herein, the C-terminus of the ybbR13 peptide and not the N-terminus appears to be important for Sfp recognition. For example, the N- terminal extensions of the ybbR13 peptide (e.g., ybbRl 1 and ybbR12) did not significantly affect the activity of this peptide as a substrate of Sfp. In contrast, when C- terminal truncations were made by removing three or five amino acid residues from the C-terminus (e.g., ybbR14 and ybbR8), such peptides failed to be substrates of Sfp, suggesting that the C-terminal sequence of the ybbR tag is important for recognition by phosphopantetheinyl transferases such as, for example, Sfp.
[0051] As further described in the Examples, the ybbR13 peptide (SEQ ID NO: 2) had a Km of 122.8 μM for Sfp-catalyzed reaction, which is about thirty fold higher than that for PCP (4.1 μM); however, the kcat values for the ybbR peptide and PCP were similar at saturating concentrations of biotin - CoA. Sfp was able to transfer different types of small molecules from CoA to which they were conjugated, to the ybbR peptide. For example, Sfp catalyzed transfer of both fluorescein and biotin from CoA to the ybbR peptide at similar rates. The ybbR13 peptide is shown herein, to be versatile with respect to its location, when used as a substrate in a phosphopantetheinylation reaction. For example, the ybbR13 peptide is recognized by Sfp, when fused either to the N- terminus, the C-terminus, or when located anywhere between the N- and the C-termini of various proteins. Further, unlike long reaction times associated with many existing protein-labeling methodologies, the site specific labeling reaction of the ybbR tagged proteins can be carried out in cell lysates in just 10-20 minutes.
[0052] As supported and inferred by the data presented in the Examples herein, the use of the various peptides described herein including, for example, ybbR, S6 and Al, as efficient substrates of phosphopantetheinyl transferases such as Sfp and Acps, is
associated with their tendency to adopt an α-helical conformation, for example, either in
solution, or upon interaction with an enzyme having phosphopantetheinyl transferase activity.
[0053] For example, it is noted that all ybbR peptides that were good substrates for Sfp
(e.g., ybbRl 1, 12 and 13) showed high contents of α-helical conformation in 30% TFE
as measured by CD spectra, while the ybbR peptides that were poor substrates for Sfp or were not Sfp substrates, e.g., the C-terminal truncated peptides ybbR3, ybbR8 and ybbR14, showed poor α-helical formation. In fact, it has been previously reported that
in the case of the known substrates of Sfp, e.g., PCP and ACP, the amino acid residue that is modified by Sfp (i.e., the Ppant modified Serine) is immediately followed by a helix II, which is important for Sfp recognition. (Finking et al., Biochem., 43: 8946-56, 2004; Mofid et al, Biochem., 43: 4128-36, 2004; Mofid et al., J. Biol. Chem., 277:17023-31, 2002). However, when peptides TycC3 and PksLl encompassing the Ppant modified Ser and the helix II region of the known PCP and ACP were tested for Sfp catalyzed biotin - CoA modification, they were found to not be substrates of Sfp. Therefore, it appears that alpha-helical conformation alone is not sufficient for recognition by Sfp but the peptide sequences flanking the Ppant modified Serine residue in PCP and ACP need to be present for recognition by Sfp.
[0054] A helical wheel representation of the ybbR peptide sequence SLEFIASKLA
(SEQ ID NO:4) revealed that the proposed α-helix adopted by the ybbR peptide has an
amphiphilic distribution of the side chains with nonpolar residues He, Leu and Ala amino acid residues on one side of the helix and charged or polar residues Lys, GIu and Ser on the opposite side, as shown in Figure 5b. The corresponding helical wheel plots of the helix II region of the TycC3-PCP and FrenN-ACP with known NMR structures (shown in Figure 5b) showed no significant similarities with the ybbR peptide with respect to the distribution or alignment of residues on a specific side of the helix. Thus, the amphiphilic nature of the ybbR peptides appears to be unique to these substrates of phosphopantetheinyl transferases, as compared to the other known substrates. [0055] The Examples discussed herein show that the ybbR peptide could be inserted in the middle of a protein, e.g., EGFP, and still be labeled with various fluorophores using a Sfp catalyzed reaction. In view of the data presented herein, it is contemplated that the Sfp-catalyzed posttranslational modification of the ybbR peptide can be used widely for versatile site-specific protein labeling and has many advantages over the existing methodologies. For example, the size of the ybbR13 peptide (i.e., 11 residues) is much smaller compared to the size of PCP or ACP (e.g., 75-80 residues), and is less likely to interfere with the conformation and structure of most proteins.
[0056] Additionally, because of the recognition herein of the association of a α-helical
conformation by the ybbR peptides and their ability to be Sfp substrates, it is
contemplated that at least those ybbR peptides which form an α-helical conformation
without requiring any additional C-terminal amino acid sequences (e.g., at least ybbRll- 13) can also be used for the labeling of non-proteinaceous molecules. [0057] When labeling proteins, for example, an 11 amino acid residue ybbR peptide (e.g., ybbR13 or a variant thereof) can be attached to the N-terminus, the C-terminus, or in between the N- and C-terminus of a protein and be used for site specific labeling of the protein via Sfp-catalyzed transfer of a small molecule, for example, from a donor molecule, e.g, CoA. Further, a ybbR sequence as short as 5 amino acid residues shown in SEQ ID NO:1 (i.e., DSLXX, where X is any alpha helix favoring amino acid) can be used as a substrate of Sfp, when fused to the N-terminus of a target protein. Because of
the association of the ybbR peptides which adopt an α-helical conformation, either alone or when linked to a different amino acid sequence, with their ability to function as substrates in a phosphopantetheinylation reaction, these peptides are referred to herein as alpha-helical oligopeptide moieties.
[0058] The present invention also provides additional peptides such as, for example, the S series of peptides including, e.g., S6 and the A series of peptides including, e.g., Al, which can be fused to target proteins at either or both the N- and the C- terminals or anywhere between the N and C-termini, and be efficiently posttranslationally labeled with small molecules such as Ppant-fluorophores or Ppant-biotin by Sip and Acps, respectively, using various small molecule-CoA conjugates as the donors. [0059] As demonstrated in the Examples herein, S6 and Al show significant orthogonality in reactivity with Sfp and Acps. For example, as demonstrated in the Examples discussed herein, the catalytic efficiency (kcat /Km) for Sfp-catalyzed S6 labeling is more than 440-fold higher than the Acp-catalyzed S6 labeling and conversely, the specific activity (kcat /Km ) for Acps-catalyzed Al labeling is more than 30-fold higher than the Acps-catalyzed S6 labeling. Also, GFP proteins fused with the S6 tag or the Al tag are labeled with biotinyl-pantetheine with high efficiency only by Sfp or Acps, respectively. The cross-labeling of S- tagged GFP by Acps and Al -tagged GFP by Sfp is at least 25-fold lower than the labeling of the S6 and Al tagged proteins by the respective PPTase, e.g., Sfp for the S6 peptide and Acps for the Al peptide. [0060] Accordingly, a ybbR/Sfp or S6/Sφ pair in combination with a Al/Acps pair can be used for either simultaneous or sequential labeling of two target proteins with different small molecule probes, with very little or no cross labeling. The S6 peptide, however, appears to be a more efficient substrate of Sfp, relative to ybbR13, as demonstrated by the ratio of catalytic efficiencies (kcat/Km ) for Sfp- over Acps-catalyzed S6 labeling, which is 442 -fold compared to 28-fold for ybbR13 labeling. [0061] The Examples herein also demonstrate that the S6- or Al -tagged cell surface proteins including, but not limited to, for example, transferrin receptors, can be posttranslationally labeled with Sfp and Acps respectively. Due to the multiple negative charges on the ATP moiety of CoASH, CoA-conjugated small molecule probes are not membrane permeable. One advantage of the inability of the CoA - fluorescent dye conjugate to penetrate the membrane is that the intracellular background fluorescence is very low after extracellular labeling of the cell with the conjugate. This feature allows high-contrast imaging of the intracellular trafficking of internalized cell surface receptors.
[0062] Accordingly, the present invention provides short peptide tags, e.g., ybbR13, S6 and Al, which can be used for the site-specific posttranslational labeling of target proteins in reactions catalyzed by a phosphopantetheinyl transferase such as, for example, Sfp or Acps. Additionally, a pairing of ybbR13/Sfp or S6/Sfp and Al/Acps can also be used for orthogonal site-specific labeling of two target proteins, e.g., in cell lysates or on the surface of live cells.
[0063] The small size of the peptide tags, e.g., ybbR13, S6 and Al peptides compared to the full length PCP and ACP domains (80-100 residues), the versatility of the peptides for fusion to target proteins at N- or C-termmi, the structural diversities of the small molecule probes for Sfp- and Acps-catalyzed peptide modification, and the high efficiency and specificity of Sfp and Acps for the different peptides provide, a powerful protein labeling method that allows for specific orthogonal labeling of different target proteins on cell surfaces or in cell lysates.
[0064] Accordingly, in some embodiments, this invention provides a method of labeling any proteinaceous or non-proteinaceous molecule (referred to herein as a "second agent") by: (a) linking the molecule to an alpha-helical oligopeptide moiety having an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40 or SEQ ID NO:41, or a variant thereof; and (b) contacting the molecule with a desirable label (referred to herein as a "first agent") linked to a donor molecule (e.g., CoA or an analog thereof) via a linking group (e.g., the phosphopantetheine group or analog thereof of the CoA donor molecule), in the presence of a phosphopantetheinyl transferase such as, for example, Sfp or Acps, which transfers the label along with the linking group (i.e., "transferred moiety") from the donor molecule to the alpha helical oligopeptide moiety, thereby resulting in a labeled molecule. Phosphopantetheinyl transferases such as, for example, Sfp and Acps, have broad substrate specificities with respect to the first agents that may be transferred to an alpha-helical oligopeptide moiety, as described herein. For example, various first agents conjugated to CoA such as sugars, affinity probes such as biotin, glutathione, fluorescent probes such as fluorescein, Alexa Fluor dyes and redox probes such as porphyrin can be transferred to an alpha-helical oligopeptide moiety using a phosphopantetheinylation reaction. Furthermore Sfp catalyzed ybbR peptide or S6 peptide, and Acps catalyzed Al peptide labeling by various small molecule moieties is highly specific and efficient and can be accomplished in a single step. [0065] In other embodiments, this invention provides a method of labeling a proteinaceous molecule, such as, for example, a protein (i.e., a second agent), by linking an alpha-helical oligopeptide moiety to either the N-terminus of the protein (e.g., an alpha-helical oligopeptide moiety having an amino acid sequence set forth ejther in SEQ ID NO:1 or 2 or 40 or 41, or a variant thereof), the C-terminus of the protein (e.g., an alpha-helical oligopeptide moiety having an amino acid sequence set forth in SEQ ID NO:2 or 40 or 41, or a variant thereof), or anywhere between the N- and C- termini of the protein (e.g., an alpha-helical oligopeptide moiety having an amino acid sequence set forth in SEQ ID NO:2 or 40 or 41, or a variant thereof), and contacting the protein with a desirable label (i.e., a first agent) linked to a donor molecule via a linking group, in the presence of a phosphopantetheinyl transferase, e.g., Sfp or Acps, thereby resulting in a labeled protein.
[0066] For example, in some embodiments, this invention provides methods for labeling a target protein comprising contacting a target protein with a first agent, and allowing the first agent to be conjugated to the target protein via an alpha-helical oligopeptide moiety, in the presence of a phosphopantetheinyl transferase, where the target protein is a fusion of the alpha-helical oligopeptide moiety and another polypeptide.
[0067] In some embodiments, the target protein includes the alpha-helical oligopeptide moiety fused to the N-terminus of a polypeptide, where the alpha-helical oligopeptide moiety has an amino acid sequence set forth in SEQ ID NO:1 or a variant thereof. In other embodiments, the target protein includes the alpha-helical oligopeptide moiety fused to the N-terminus, the C-terminus, or anywhere between the N-terminus and the C-terminus of a polypeptide, where the alpha-helical oligopeptide moiety has an amino acid sequence set forth in SEQ ID NO:2 or a variant thereof. In yet other embodiments, the target protein includes at least two alpha-helical oligopeptide moieties, a first alpha- helical oligopeptide moiety having an amino acid sequence set forth in SEQ ID NO:1 or a variant thereof at the N-terminus of a polypeptide and one or more of an alpha-helical oligopeptide moiety having an amino acid sequence set forth in SEQ ID NO:2, or a variant thereof either the C-terminus of the polypeptide or located anywhere between the N- and the C-termini.
[0068] In some embodiments, the target protein includes at least one alpha-helical oligopeptide moiety fused to the N-terminus, the C-terminus, or anywhere between the N-terminus and the C-terminus of a polypeptide, where the alpha-helical oligopeptide moiety has an amino acid sequence set forth in SEQ ID NO:40 or a variant thereof. In yet other embodiments, the target protein includes at least one alpha oligopeptide moiety fused to the N-terminus, the C-terminus, or anywhere between the N-terminus and the C-terminus of a target protein, where the alpha-oligopeptide moiety has an amino acid sequence set forth in SEQ ID NO:415 or a variant thereof.
[0069] In further embodiments, two separate target proteins may be labeled using the methods of the present invention. For example, a first target protein can include one or more alpha-helical oligopeptide moieties including the amino acid sequences set forth in SEQ ID NO:2 or SEQ ID NO:40, or a variant thereof, and a second target protein can include an alpha-helical oligopeptide moiety including an amino acid sequence set forth in SEQ ID NO:41, or a variant thereof, either at the N-terminus, the C-terminus or anywhere between the N- and the C-termini. Accordingly, the first target protein including at least one alpha helical oligopeptide moiety having an amino acid sequence selected from SEQ ID NO:2 or 40, can be labeled using an Sfp catalyzed reaction, and the second target protein including at least one alpha helical oligopeptide moiety having an amino acid sequence set forth in SEQ ID NO:41, can be labeled using an Acps catalyzed reaction, either simultaneously or sequentially.
[0070] In methods described herein, a first agent along with a linking group {i.e., the transferred moiety) is transferred to a conserved serine residue on an alpha-helical . oligopeptide moiety via a phosphopantetheinylation reaction. In some embodiments, the first agent is attached to a linking group and a leaving group, and then is transferred with the linking group to the alpha-helical oligopeptide moiety via a phosphopantetheinylation reaction. A schematic of such a reaction is shown below:
Figure imgf000025_0001
Figure imgf000025_0002
Leaving Group
P-Pant transferase
Figure imgf000025_0003
Figure imgf000025_0004
SCHEME 1
[0071 ] The terms "phosphopantetheinyl transferase," and "protein having phosphopantetheinyl transferase activity," as used herein, refer to any enzyme or a fragment thereof, which is capable of transferring a 4'-phosphopantetheine group from a donor molecule, such as, for example, coenzyme A (CoA) or an analog thereof, to a substrate such as, for example, an alpha-helical oligopeptide moiety. Phosphopantetheinyl transferases and proteins having phosphopantetheinyl transferase activity also include fragments, e.g., active fragments or fragments of a phosphopantetheinyl transferase or a protein having phosphopantetheinyl transferase activity, which are capable of transferring a transferred moiety (e.g., a first agent linked to a linking group) from a donor molecule such as, for example, Mq-L-N, to a substrate. Phosphopantetheinyl transferases (PPTases) are enzymes which catalyze post- translational modification of carrier proteins associated with fatty acid synthetases (FASs), polyketide synthetases (PKSs) and non-ribosomal polypeptide synthetases (NPRs). Phosphopantetheinyl transferases have been classified into three groups, based on sequence and structural similarity and substrate specificity. Members of the first group, for example, Acps of Escherichia coli, are about 120 amino acid residues long, function as homotrimers, and have fairly narrow substrate specificities limited to, for example, ACPs of type II FAS and PKS systems. Members of the second group, exemplified by Sφ of Bacillus subtilis, are usually about 240 amino acid residues long, function as monomers, and have been reported to have broad substrate specificities that include carrier proteins associated with NRPs, FASs and PKSs. (Suo et ah, Proc. Natl. Acad. Sci. USA3 98:99-104, 2001; Quadri et ah, Biochem., 37:1585-95, 1998; Liu et ah, Arch. Microbiol, 183:37-44, 2005). The third group includes PPTases that are attached covalently to the type I FASs, such as those associated with the yeast cytosolic FAS. (Fichtlscherer et ah, Eur. J. Biochem., 267:2666-71, 2000).
[0072] Phosphopantetheinyl transferases encompassed by this invention include both naturally occurring proteins having phosphopantetheinyl transferase activity including, but not limited to, Acps from E. Coli and Sfp from B. subtilis, fatty acid synthases (FAS) from S. cerevisiae, S. pombe, C. albacans, E. nidulans, and P. patulum, EntD from E. Coli, S.flexneri, S. typhimurium and S. austin, Psf-1 from B. pumilus, Gsp from B. brevis, Hetl from Anabaena sp., Lys5 from S. cerevisiae, Lpa-14 from B. subtilis and 0195 from E. coli, and homologs thereof. The term "homolog" of a phosphopantetheinyl transferase is intended to include phosphopantetheinyl transferases from a species other than the ones described, which are capable of phospopantetheinylating an alpha-helical oligopeptide moiety described herein. For example, in some embodiments, a homolog of Sφ includes a phosphopantetheinyl transferase from a species other than B. subtilis, which is capable of phosphopantetheinylating an alpha-helical oligopeptide moiety including an amino acid sequence of DSLXX (SEQ ID NO:1) or DSLXXX (SEQ ID NO:39) or GDSLSWLLRLLN (SEQ ID NO:40) when linked to the N-terminus of any polypeptide, where X is an alpha-helix favoring amino acid. In another embodiment, a homolog of Sfp includes a phosphopantetheinyl transferase from a species other than B. subtilis, which is capable of phosphopantetheinylating an alpha-helical oligopeptide moiety including an amino acid sequence set forth in SEQ ID NO:2, or SEQ ID NO:40, or a variant thereof, independent of what it is linked to. In yet another embodiment, a homolog of Acps includes a phosphopantetheinyl transferase from a species other than E. CoIi, which is capable of phosphopantetheinylating an alpha-helical oligopeptide moiety including an amino acid sequence of GDSLDMLEWSLM (SEQ ID NO:41), or a variant thereof.
[0073] Homologs of phosphopantetheinyl transferases used in the methods described herein, for example, Sfp of B. subtilis, and Acps from E. CoIi, can vary in the degree of amino acid sequence homology or identity with the amino acid sequence of Sfp or Acps, so long as the homolog enzyme is capable of phosphopantetheinylating a substrate as described herein. Homologs of Sfp and Acps within the scope of this invention can be encoded by a nucleic acid having any degree of nucleic acid sequence identity with a nucleic acid encoding B. subtilis Sfp (Grossman et al., J. Bacteriol., 175(19):6203-6211, 1993)).
[0074] Homology, also termed herein "identity" refers to sequence similarity between two proteins (peptides) or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same nucleotide base or amino acid, then the molecules are homologous, or identical, at that position. A degree (or percentage) of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A degree or percentage of "identity" between amino acid sequences refers to amino acid sequence similarity wherein conserved amino acids are considered to be identical for the purpose of determining the degree or percentage of similarity. A conserved amino acid substitution is, e.g., substitution of one amino acid having a negative side chain for another amino acid having a negative side chain.
[0075] In some embodiments, homologs of Sfp, Acps, or other members of the phosphopantetheine transferase superfamily, have an overall amino acid sequence identity or similarity of at least about 50%, at least about 60%, at least about 70 %, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater than 99% with Sfp used in the methods described herein, wherein the homologs are capable of phosphopantetheinylating an alpha-helical oligopeptide moiety, as described herein. [0076] Also encompassed by this invention are enzymes that are recombinantly or synthetically produced, and fragments thereof having phosphopantetheinyl transferase activity. Examples of various members of the phosphopantetheinyl transferase family can be found, for example, in Lambalot et al., Chem. & Biol., 3:923-926, 1996 and in U.S. Patent No. 6,579,695, the entire contents of which are incorporated by reference herein.
[0077] Phosphopantetheinylation occurs by transfer of the 4'-phosphopantetheine (P- pant) prosthetic group from coenzyme A to a conserved serine residue in carrier proteins, which converts the carrier proteins from their inactive "apo" form to their active "holo" form. (Id. at Lambalot et al).
[0078] The phosphopantetheinyl transferase, Sfp, activates NRPS and PKS clusters in B. subtilis by posttranslational transfer of the Ppant group from CoA to a conserved serine residue in each of the ACPs and PCPs associated with the biosynthesis of NRPS and PKS. It has been previously reported that the Ppant-modified Serine (underlined) in the PCPs is located in a conserved motif within the sequence DxFFxxLGG(H/D)S(L/I) (SEQ ID NO:5) ("x" denotes any of the 20 proteinogenic amino acids and "H/D" denotes either H or D at the same position). However a nineteen residue peptide including the sequence GVTDNFFMIGGHSLKAMMM (SEQ ID NO:6) from the B. subtilis SrfBl-PCP encompassing the conserved motif was previously tested for Sfp catalyzed Ppant modification and was found not to be a substrate of Sfp. (Id. at Quadri et al.)
[0079] The terms "isolated," and "purified," as used in the context of a phosphopantetheinyl transferase or a protein having phosphopantetheinyl transferase activity, e.g., Sfp and Acps, refer to enzymes or fragments thereof that are substantially free of cellular material when purified, for example, from a cell which naturally expresses such an enzyme or fragment, or culture medium when purified from recombinant cells, or chemicals when produced synthetically. [0080] Cells from which a phosphopantetheinyl transferase or a fragment thereof having phosphopantetheinyl transferase activity is isolated may either be prokaryotic or eukaryotic cells, including but not limited to, for example, yeast cells, plant cells, bacterial cells, insect cells and mammalian cells. Also encompassed by this disclosure are recombinant enzymes that may be "isolated" or "purified" from a cell that has been genetically engineered or modified to produce a phosphopantetheinyl transferase or fragment thereof having phosphopantetheinyl transferase activity. [0081] If a phosphopantetheinyl transferase used in the methods described herein is produced recombinantly or synthetically, it may not be necessary to purify the enzyme significantly. A recombinantly produced phosphopantetheinyl transferase may have an activity identical to that of its natural counterpart, or it may have an activity which varies more or less from that of its natural counterpart. For example, a recombinantly produced phosphopantetheinyl transferase can vary in the efficiency of catalysis. It is possible to modify the amino acid sequence of the enzyme to, e.g., improve its efficiency, or to change its substrate specificity, or both.
[0082] Purification of a phosphopantetheinyl transferase or a protein or fragment thereof having phosphopantetheinyl transferase activity can be achieved using standard procedures well-known in the art. Exemplary procedures include, for example, affinity purification using a column containing a known substrate of a phosphopantetheinyl transferase enzyme such as, an apo-acyl carrier protein, or an alpha-helical oligopeptide moiety, as described herein. In some embodiments, a procedure used for purification of a phosphopantetheinyl transferase or a protein having phosphopantetheinyl transferase activity or a fragment thereof, results in purification of a phosphopantetheinyl transferase or a protein or fragment having phosphopantetheinyl transferase activity to at least 50% purity, or at least 60% purity, or at least 70% purity, or at least 80% purity, or at least 90% purity, or at least 95% purity, or at least 96% purity, or at least 97% purity, or at least 98% purity, or at least 99% purity, or greater than 99% purity. In some embodiments, purification of a phosphopantetheinyl transferase or a protein having phosphopantetheinyl transferase activity or a fragment thereof results in an enrichment of phosphopantetheinyl transferase activity by at least about 500 fold, or at least 600 fold, or at least 700 fold, or at least 800 fold, or at least 900 fold, or at least 1000 fold, or at least 5000 fold, or at least 10,000 fold, or at least 50,000 fold, or at least 70,000 fold, or greater than 70,000 fold. Exemplary methods of purification are described in, for example, U.S. Patent No. 6,579,695, the entire content of which is incorporated by reference herein. [0083] In some embodiments, a purified phosphopantetheinyl transferase or an enzyme having phosphopantetheinyl transferase activity has a specific activity of at least 100 mU/mg, or at least 200 mU/mg, or at least 250 mU/mg, or at least 300 mU/mg, or at least 400 mU/mg, or at least 500 mU/mg, or greater.
[0084] The term "first agent," as used herein, refers to any entity, biological or synthetic in nature, which can be transferred from a donor molecule to an alpha-helical oligopeptide moiety. In some methods described herein, a first agent is linked to a donor molecule such as, for example, coenzyme A, via a linking group, and is transferred along with the linking group or a part thereof, to an alpha-helical oligopeptide moiety. In some embodiments, the first agent is linked to the donor molecule via a covalent bond. In some of the methods described herein, the first agent along with the linking group (i.e., transferred moiety), is transferred from the donor molecule to an alpha-helical oligopeptide moiety linked to a second agent using a phosphopantetheinylation reaction.
[0085] Examples of first agents include, but are not limited to, for example, small molecules moieties (e.g., drag moieties, toxins), protons, haptens, affinity probes, spectroscopic probes, radioactive probes, peptides, non-naturally occurring amino acids, nucleic acids, lipid molecules, radical generating molecules, singlet oxygen generating molecules, polymers, sugars (e.g., monosaccharides, disaccharides, polysaccharides, and other carbohydrate containing moieties), antibodies and antibody fragments, enzymes, enzyme substrates, chelating agents and receptor binding molecules. [0086] The term "spectroscopic probes" includes, but is not limited to, moieties which can be detected using spectroscopic techniques. Examples of spectroscopic probes include, but are not limited to, fluorophores (e.g., Fluorescein), chromophores (e.g., luminal, luciferase, luciferin, and aequorin), magnetic probes and contrast reagents (e.g., MRI contrast reagents). Other examples of spectroscopic probes include, but are not limited to, phosphorescent probes and PET labels.
[0087] The term "affinity probe" includes, but is not limited to, moieties which can be used to bind to an affinity matrix, e.g., to enhance purification of molecules of the invention. Examples of affinity probes include biotin and glutathione and analogs thereof. Examples of biotin analogs include, for example, an N-ketone biotin analog, a ketone biotin analog, an N-azide biotin analog, an azide biotin analog, an N-acyl azide biotin analog, an NBD-GABA biotin analog, a 1,2-diamine biotin analog, an N-alkyne biotin analog and a tetrathiol biotin analog.
[0088] The term "affinity matrix" includes a matrix, such as agarose, controlled pore glass, or poly (styrenedivinyl) benzene to which an affinity ligand is attached. The affinity ligand binds to the affinity probe and the contaminating molecules are not bound to the affinity ligand. The molecule of the invention with the affinity probe can be eluted from the affinity matrix using known protocols.
[0089] The term "antibody," as used herein, includes monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispeciflc antibodies (e.g., bispecifc antibodies), chimeric antibodies, CDR-grafted antibodies, humanized antibodies, human antibodies and antigen-binding fragments thereof, for example, an antibody light chain (VL), an antibody heavy chain (VH), a single chain antibody (scFv), a F(ab')2 fragment, a Fab fragment, an Fd fragment, an Fv fragment, and a single domain antibody fragment (DAb). Examples of antibodies including, for example, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, fully human antibodies, antibodies produced in transgenic organisms, synthetic antibodies, single domain antibodies, antibodies with modified Fc regions, and camelid like antibodies are further described in, e.g., 6,331,415, 4,816,567; 5,225,539; 6,180,370; 5,693,762; 5,693,761; 5,585,089; 6,548,640; 5,859,205; 6,632,927; 6,407,213; 6,639,055; 5,885,793; 6,150,584; 5,770,429; 6,300,064; 6,846,634; 6,737,056; and 6,670,453, the entire contents of which are incorporated by reference herein.
[0090] The term "receptor binding molecule," as used herein includes an agonist, an antagonist or a ligand of a receptor, or analogs thereof. A ligand may either be a natural ligand to which a receptor binds, or a molecule which is a functional analog of the natural ligand. The functional analog may be a ligand with structural modifications, or may be a wholly unrelated molecule which has a molecular shape which interacts with the appropriate ligand binding determinants. Ligands may serve as agonists or antagonists, see, e.g., Goodman, et al. (eds. 1990) Goodman & Gilman's: The Pharmacological Bases of Therapeutics, Pergamon Press, New York. [0091] Examples of "radioactive probes" include radionuclides. Examples of radionuclides include 123Iodine, 125Iodine, 131Iodine, 105Rhodium, 67Gallium, 153Sm, 177Lu, 186Re, 188Re, 166Ho, 67Cu, 90Y, l "indium, 18Fluorine, or """Technetium (Tc99m). Radionuclides for indirect labeling include, for example, 111In and 90Y. In some
embodiments, the radionuclides used typically produce high energy α- or β-particles which have a short path length. In some embodiments, such radionuclides can be conjugated using methods described herein, to a molecule which would target them specifically to neoplastic cells, for example, thereby killing such cells. [0092] Examples of chelating agents include, but are not limited to, 1- isothiocycmatobenzyl-3-methyldiothelene triaminepentaacetic acid ("MX-DTPA") and cyclohexyl diethylenetriamine pentaacetic acid ("CHX-DTPA") derivatives. Other chelating agents include, for example, P-DOTA and EDTA derivatives, and EGTA. [0093] Additional examples of first agents include non-naturally occurring amino acids. Examples of non-naturally occurring amino acids include for glutamine (GIu) or glutamic acid residues: α-aminoadipate molecules; for tyrosine (Tyr) residues: phenylalanine (Phe), 4-carboxymethyl-Phe, pentafluorophenylalanine (PfPhe), 4- carboxymethyl-L-phenylalanine (cmPhe), 4-carboxydifluoromethyl-L-phenylalanine (F2cmPhe), 4-phosphonomethyl-phenylalanine (Pmp), (difluorophosphonomethyl) phenylalanine (F2Pmp), O-malonyl-L-tyrosine (malTyr or OMT), and fluoro-O- malonyltyrosine (FOMT); for proline residues: 2-azetidinecarboxylic acid or pipecolic acid (which have 6-membered, and 4-membered ring structures respectively); 1- aminocyclohexylcarboxylic acid (Ac6c); 3-(2-hydroxynaphtalen-l-yl)-propyl; S- ethylisothiourea; 2-NH2-thiazoline; 2-NH2-thiazole; asparagine residues substituted with 3-indolyl-propyl at the C terminal carboxyl group. Modifications of cysteines, histidines, lysines, arginines, tyrosines, glutamines, asparagines, prolines, and carboxyl groups are known in the art and are described in U.S. Patent No. 6,037,134, the entire contents of which are incorporated herein by reference.
[0094] Also encompassed by this invention is the use of enzymes or enzyme substrates as first agents. Examples of these include (enzyme (substrate)): Alkaline Phosphatase (4-Methylumbelliferyl phosphate Disodium salt; 3-Phenylumbelliferyl phosphate Hemipyridine salt); Aminopeptidase (L-Alanine-4-methyl-7-coumar- inylamide trifluoroacetate; Z-L-argimne-4-methyl-7-coumarinylamide hydrochloride; Z-glycyl-L- proline-4-methyl-7-coumarinylamide); Aminopeptidase B (L-Leucine-4-methyl-7- coumarinylamide hydrochloride); Aminopeptidase M (L-Phenylalanine 4-methyl-7- coumarinylamide trifluoroacetate); Butyrate esterase (4-Methylumbelliferyl butyrate); Cellulase (2-Chloro-4-nitrophenyl-beta-D-cellobioside); Cholinesterase (7-Acetoxy-l- methylquinolinium iodide; Resorufin butyrate); alpha-Chymotrypsin, (Glutaryl-L- phenylalanine 4-methyl-7-coumarinylamide); N-(N-Glutaryl-L-phenylalanyl)-2- aminoacridone; N-(N-Succinyl-L-phenylala- nyl)-2-aminoacridone); Cytochrome P450 2B6 (7-Ethoxycoumarin); Cytosolic Aldehyde Dehydrogenase (Esterase Activity) (Resorufm acetate); Dealkylase (O7-Pentylresorufm); Dopamine beta-hydroxylase (Tyramine); Esterase (8-Acetoxypyrene-l,3,6-trisulfonic acid Trisodium salt; 3-(2 Benzoxazolyl) umbelliferyl acetate; S-Butyryloxypyrene-l^ό-trisulfonicacid Trisodium salt; 2',7?-Dichlorofluorescin diacetate; Fluorescein dibutyrate; Fluorescein dilaurate; 4- Methylumbelliferyl acetate; 4-Methylumbelliferyl butyrate; 8-Octanoyloxypyrene-l,3,6- trisulfonic acid Trisodium salt; 8-Oleoyloxypyrene-l,3,6-trisulfonic acid Trisodium salt; Resorufϊn acetate); Factor X Activated (Xa) (4-Methylumbelliferyl 4-guanidinobenzoate hydrochloride Monohydrate); Fucosidase, alpha-L-( 4-Methylumbelliferyl-alpha-L- fucopyranoside); Galactosidase, alpha-(4-Methylumbelliferyl-alpha-D galactopyranoside); Galactosidase, beta- (6,8-Difluoro-4-methylumbelliferyl-beta-D- galactopyranoside; Fluorescein di(beta-D-galactopyranoside); 4-Methylumbelliferyl- alpha-D-galactopyranoside^-Methylumbelliferyl-beta-D-lactoside: Resorafin-beta-D- galactopyranoside; 4-(Trifluoromethyl)umbelliferyl-beta-D-galactopyranoside; 2- Chloro-4-nitrophenyl-beta-D-lactoside); Glucosaminidase, N-acetyl-beta- (4- Methylumbelliferyl-N-acetyl-beta-D-glucosaminide Dihydrate); Glucosidase, alpha-(4- Methylumbelliferyl-alpha-D-glucopyranoside); Glucosidase, beta- (2-Chloro-4- nitrophenyl-beta-D-glucopyranoside; 6,8-Difluoro-4-methylumbelliferyl-beta-D- glucopyranoside^-Methylumbelliferyl-beta-D-glucopyranosidej Resorufln-beta-D- glucopyranoside; 4-(Trifluoromethyl) umbelliferyl-beta-D-glucopyranoside); Glucuronidase, beta-(6,8-Difluoro-4-methylumbelliferyl-beta-D-glucuronide Lithium salt; 4-Methylumbelliferyl-beta-D-glucuronide Trihydrate); Leucine aminopeptidase( L- Leucine-4-methyl-7-coumarinylamide hydrochloride); Lipase (Fluorescein dibutyrate; Fluorescein dilaurate; 4-Methylumbelliferyl butyrate; 4-Methylumbelliferyl enanthate; 4-Methylumbelliferyl oleate; 4-Methylumbelliferyl palmitate; Resorufm butyrate); Lysozyme (4-Methylumbelliferyl-N,N',N'-triacetyl-beta-chitotrioside); Mannosidase, alpha- (4-Methylumbelliferyl -alpha-D-mannopyranoside); Monoamine oxidase (Tyramine); Monooxygenase (7-Ethoxycoumarin); Neuraminidase (4- Methylumbelliferyl-N-acetyl-alpha-D-neuraminic acid Sodium salt Dihydrate); Papain (Z-L-argmme-4-methyl-7-coumarinylamide hydrochloride); Peroxidase (Dihydrorhodamine 123); Phosphodiesterase (1-Naphthyl 4-phenylazophenyl phosphate; 2-Naphthyl 4-phenylazophenyl phosphate); Prolyl endopeptidase (Z-glycyl-L-proline-4- methyl-7-coumarinylamide; Z-glycyl-L-proline-2-naphthylamide; Z-glycyl-L-prolme-4- nitroanilide); Sulfatase (4-Methylumbelliferyl sulfate Potassium salt); Thrombin (4- Methylumbelliferyl 4-guanidinobenzoate hydrochloride Monohydrate); Trypsin (Z-L- arginine-4-methyl-7-coumarinylamide hydrochloride; 4-Methylumbelliferyl 4- guanidinobenzoate hydrochloride Monohydrate); Tyramine dehydrogenase (Tyramine). [0095] The term "small molecule" include moieties with molecular weights between lτ 10,000 Daltons. In a further embodiment, the small molecules are drug moieties or other agents with desirable biological properties.
[0096] The term "drug moiety" includes agents which would be beneficial to the subject. A "drug moiety," as used herein, includes any agent that can be used either to treat an actual cause of the disorder or condition being treated in a subject and/or to provide relief to the patients from symptoms of a disorder. Drug moieties may include, but are not limited to, anti-inflammatory, anticancer, cytotoxic, antiinfective (e.g., antifungal, antibacterial, anti-parasitic, anti- viral, etc.), and anesthetic agents. [0097] Other examples of drug moieties encompassed by this invention include anabolic agents, antacids, anti-asthmatic agents, anti-cholesterolemic and anti-lipid agents, anti-coagulants, anti-convulsants, anti-diarrheals, anti-emetics, anti-infective agents, anti-inflammatory agents, anti-manic agents, anti-nauseants, anti-neoplastic agents, anti-obesity agents, anti-pyretic and analgesic agents, anti-spasmodic agents; anti-thrombotic agents, anti-uricemic agents, anti-anginal agents, antihistamines, antitussives, appetite suppressants, cerebral dilators, coronary dilators, decongestants, diuretics, erythropoietic agents, expectorants, gastrointestinal sedatives, hyperglycemic agents, hypnotics, hypoglycemic agents, laxatives, mucolytic agents, neuromuscular drugs, peripheral vasodilators, psychotropics, sedatives, stimulants, thyroid and antithyroid agents, uterine relaxants, vitamins, and prodrugs.
[0098] Specific examples of drug moieties include: anti-neoplasties such as androgen inhibitors, antimetabolites, cytotoxic agents, and immunomodulators; anti-tussives such as dextromethorphan, dextromethorphan hydrobromide, noscapine, carbetapentane citrate, and chlorphedianol hydrochloride; antihistamines such as chlorpheniramine maleate, phenindamine tartrate, pyrilamine maleate, doxylamine succinate, and phenyltoloxamine citrate; decongestants such as phenylephrine hydrochloride, phenylpropanolamine hydrochloride, pseudoephedrine hydrochloride, and ephedrine; various alkaloids such as codeine phosphate, codeine sulfate and morphine; antiarrhythmics such as N-acetylprocainamide; antipyretics and analgesics such as acetaminophen, aspirin and ibuprofen; appetite suppressants such as phenylpropanolamine hydrochloride or caffeine; expectorants such as guaifenesin; and anti- infective agents such as antifungals, anti-virals, antiseptics and antibiotics. [0099] Other examples of drug moieties include analgesics, such as nonsteroidal antiinflammatory drugs, opiate agonists and salicylates; antihistamines, such as H1 -blockers and H2 -blockers; anti-infective agents, such as anthelmintics, antianaerobics, antibiotics, aminoglycoside antibiotics, antifungal antibiotics, cephalosporin antibiotics,
macrolide antibiotics, miscellaneous β-lactam antibiotics, penicillin antibiotics,
quinolone antibiotics, sulfonamide antibiotics, tetracycline antibiotics, antimycobacterials, antituberculosis antimycobacterials, antiprotozoals, antimalarial antiprotozoals, antiviral agents, anti-retroviral agents, scabicides, and urinary anti- infectives; antineoplastic agents, such as alkylating agents, nitrogen mustard aklylating agents, nitrosourea alkylating agents, antimetabolites, purine analog antimetabolites, pyrimidine analog antimetabolites, hormonal antineoplastics, natural antineoplastics, antibiotic natural antineoplastics, and vinca alkaloid natural antineoplastics; autonomic agents, such as anticholinergics, antimuscarinic anticholinergics, ergot alkaloids, parasympathomimetics, cholinergic agonist parasympathomimetics, cholinesterase
inhibitor parasympathomimetics, sympatholytics, α-blocker sympatholytics, β-blocker sympatholytics, sympathomimetics, and adrenergic agonist sympathomimetics;
cardiovascular agents, such as antianginals, β-blocker antianginals, calcium-channel
blocker antianginals, nitrate antianginals, antiarrhythmics, cardiac glycoside antiarrhythmics, class I antiarrhythmics, class II antiarrhythmics, class III antiarrhythmics, class IV antiarrhythmics, antihypertensive agents, .alpha. -blocker antihypertensives, angiotensin-converting enzyme inhibitor (ACE inhibitor)
antihypertensives, β-blocker antihypertensives, calcium-channel blocker antihypertensives, central-acting adrenergic antihypertensives, diuretic antihypertensive agents, peripheral vasodilator antihypertensives, antilipemics, bile acid esequestrant antilipemics, HMG-CoA reductase inhibitor antilipemics, inotropes, cardiac glycoside inotropes, and thrombolytic agents; dermatological agents, such as antihistamines, antiinflammatory agents, corticosteroid anti-inflammatory agents, antipruritics/local anesthetics, topical anti-infectives, antifungal topical anti-infectives, antiviral topical anti-infectives, and topical antineoplastics; electrolytic and renal agents, such as acidifying agents, alkalinizing agents, diuretics, carbonic anhydrase inhibitor diuretics, loop diuretics, osmotic diuretics, potassium-sparing diuretics, thiazide diuretics, electrolyte replacements, and uricosuric agents; enzymes, such as pancreatic enzymes and thrombolytic enzymes; gastrointestinal agents, such as antidiarrheals, antiemetics, gastrointestinal anti-inflammatory agents, salicylate gastrointestinal anti-inflammatory agents, antacid anti-ulcer agents, gastric acid-pump inhibitor anti-ulcer agents, gastric mucosal anti-ulcer agents, H2-blocker anti-ulcer agents, cholelitholytic agents, digestants, emetics, laxatives and stool softeners, and prokinetic agents; general anesthetics, such as inhalation anesthetics, halogenated inhalation anesthetics, intravenous anesthetics, barbiturate intravenous anesthetics, benzodiazepine intravenous anesthetics, and opiate agonist intravenous anesthetics; hematological agents, such as antianemia agents, hematopoietic antianemia agents, coagulation agents, anticoagulants, hemostatic coagulation agents, platelet inhibitor coagulation agents, thrombolytic enzyme coagulation agents, and plasma volume expanders; hormones and hormone modifiers, such as abortifacients, adrenal agents, corticosteroid adrenal agents, androgens, anti-androgens, antidiabetic agents, sulfonylurea antidiabetic agents, antihypoglycemic agents, oral contraceptives, progestin contraceptives, estrogens, fertility agents, oxytocics, parathyroid agents, pituitary hormones, progestins, antithyroid agents, thyroid hormones, and tocolytics; immunobiologic agents, such as immunoglobulins, immunosuppressives, toxoids, and vaccines; local anesthetics, such as amide local anesthetics and ester local anesthetics; musculoskeletal agents, such as anti- gout anti-inflammatory agents, corticosteroid anti-inflammatory agents, gold compound anti-inflammatory agents, immunosuppressive anti-inflammatory agents, nonsteroidal anti-inflammatory drugs (NSAJDs) (e.g., diclofenac, ibuprofen, ketoprofen, and naproxen), salicylate anti-inflammatory agents, skeletal muscle relaxants, neuromuscular blocker skeletal muscle relaxants, and reverse neuromuscular blocker skeletal muscle relaxants; neurological agents, such as anticonvulsants, barbiturate anticonvulsants, benzodiazepine anticonvulsants, anti-migraine agents, antiparkinsonian agents, anti- vertigo agents, opiate agonists, and opiate antagonists; ophthalmic agents, such as anti-
glaucoma agents, β-blocker anti-gluacoma agents, miotic anti-glaucoma agents,
mydriatics, adrenergic agonist mydriatics, antimuscarinic mydriatics, ophthalmic anesthetics, ophthalmic anti-infectives, ophthalmic aminoglycoside anti-infectives, ophthalmic macrolide anti-infectives, ophthalmic quinolone anti-infectives, ophthalmic sulfonamide anti-infectives, ophthalmic tetracycline anti-infectives, ophthalmic antiinflammatory agents, ophthalmic corticosteroid anti-inflammatory agents, and ophthalmic nonsteroidal anti-inflammatory drugs (NSAIDs); psychotropic agents, such as antidepressants, heterocyclic antidepressants, monoamine oxidase inhibitors (MAOIs), selective serotonin reuptake inhibitors (SSRIs), tricyclic antidepressants, antimanics, antipsychotics, phenothiazine antipsychotics, anxiolytics, sedatives, and hypnotics, barbiturate sedatives and hypnotics, benzodiazepine anxiolytics, sedatives, and hypnotics, and psychostimulants; respiratory agents, such as antitussives, bronchodilators, adrenergic agonist bronchodilators, antimuscarinic bronchodilators, expectorants, mucolytic agents, respiratory anti-inflammatory agents, and respiratory corticosteroid anti-inflammatory agents; toxicology agents, such as antidotes, heavy metal antagonists/chelating agents, substance abuse agents, deterrent substance abuse agents, and withdrawal substance abuse agents; and vitamins, such as vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, and vitamin K. [00100] Additional examples of drug moieties include cytotoxic drugs, for example, those which are used for cancer therapy, e.g., anticancer agents. The terms "cytotoxin," "cytotoxic agent" and "anticancer agent" means any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit or destroy a cell or malignancy. Exemplary cytotoxins include, but are not limited to, radionuclides, biotoxins, enzymatically active toxins, cytostatic or cytotoxic therapeutic agents, prodrugs, immunologically active ligands and biological response modifiers such as cytokines. Any cytotoxin that acts to retard or slow the growth of immunoreactive cells or malignant cells is within the scope of the present disclosure. [00101]Exemplary cytotoxins include, in general, cytostatic agents, alkylating agents, antimetabolites, antiproliferative agents, tubulin binding agents, hormones and hormone antagonists, and the like. Exemplary cytostatics that are compatible with the present invention include alkylating substances, such as mechlorethamine, triethylenephosphoramide, cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan or triaziquone, also nitrosourea compounds, such as carmustine, lomustine, or semustine. Other classes of cytotoxic agents include, for example, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, and the podophyllotoxins. Other members of those classes include, for example, adriamycin, carminomycin, daunorubicin (daunomycin), doxorubicin, aminopterin, methotrexate, methopterin, mithramycin, streptonigrin, dichloromethotrexate, mitomycin C, actinomycin-D, porfiromycin, 5-fluorouracil, floxuridine, florafur, 6-mercaptopurine, cytarabine, cytosine arabinoside, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine and the like. Still other cytotoxins include taxol, taxane, cytochalasin B, gramicidin D, ethidium bromide, emetine, tenoposide, colchicin, dihydroxy anthracin dione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Hormones and hormone antagonists, such as corticosteroids, e.g. prednisone, progestins, e.g. hydroxyprogesterone or medroprogesterone, estrogens, e.g. diethylstilbestrol, antiestrogens, e.g. tamoxifen, androgens, e.g. testosterone, and aromatase inhibitors, e.g. aminogluthetimide may also be used as first agents.
[00102] Other examples of drug moieties include, for example, anticancer agents such as angiogenesis inhibitors (e.g., Angiostatin Kl-3, DL-α-Difluoromethyl-ornithine, Endostatin, Fumagillin, Genistein, Minocycline, Staurosporine, and (±)-Thalidomide); DNA-intercalator or cross-linkers (e.g., Bleomycin, Carboplatin, Carmustine, Chlorambucil, Cyclophosphamide, cis-Diammineplatinum(II) dichloride (Cisplatin), Melphalan, Mitoxantrone, and Oxaliplatin); DNA synthesis inhibitors (e.g., (±)- Amethopterin (Methotrexate), 3-Amino-l,2,4-benzotriazine 1,4-dioxide, Aminopterin, Cytosine β-D-arabinofuranoside, 5-Fluoro-5'-deoxyuridine, 5-Fluorouracil, Ganciclovir, Hydroxyurea, and Mitomycin C); DNA-RNA transcription regulators (e.g., Actinomycin D, Daunorubicin, Doxorubicin, Homoharringtonine, and Idarubicin); enzyme inhibitors (e.g., S(+)-Camptothecin, Curcumin, (-)-Deguelin, 5,6-Dichlorobenz-imidazole 1-β-D- ribofuranoside, Etoposide, Formestane, Fostriecin, Hispidin, 2-Immo-l-imidazoli- dineacetic acid (Cyclocreatine), Mevinolin, Trichostatin A, Tyrphostin AG 34, and Tyrphostin AG 879); agents which regulate genes (e.g., 5-Aza-2'-deoxycytidine, 5- Azacytidine, Cholecalciferol (Vitamin D3), 4-Hydroxytamoxifen, Melatonin, Mifepristone, Raloxifene, trans-Retinal (Vitamin A aldehydes), Retinoic acid, Vitamin A acid, 9-cis-Retinoic Acid, 13-cis-Retinoic acid, Retinol (Vitamin A), Tamoxifen, and Troglitazone); microtubule inhibitors (e.g., Colchicine, Dolastatin 15, Nocodazole, Paclitaxel, Podophyllotoxin, Rhizoxin, Vinblastine, Vincristine, Vindesine, and Vinorelbine (Navelbine)); 17-(Allylamino)-17-demethoxygeldanarnycin, 4-Amino-l,8- naphthalimide, Apigenin, Brefeldin A, Cimetidine, Dichloromethylene-diphosphonic
acid, Leuprolide (Leuprorelin), Luteinizing Hormone-Releasing Hormone, Pifithrin-α,
Rapamycin, Sex hormone-binding globulin, and Thapsigargin. [00103]Examples of drug moieties further include doxorubicin, etoposide, taxane, paclitaxel, fluorouracyl, mitomycin, camptothecin, gemcitabine, geldanamycin, epothilone, cephalostatin, tubulin inhibitors, a vinca alkaloid, proteasome inhibitors, neocarzinostatin, calicheamicin, maytansinoids, (RS)-cyclophosophamide, 6- mercaptopurines, auristatin E, daunorubicin, and derivatives or analogs thereof. [00104]It is understood that the drug moieties may be attached to the linking group through any atom which allows the resulting molecule and/or drug moiety to perform its intended function. The drug moieties also include pharmaceutically acceptable prodrugs, salts, esters, amides, and ethers of the drug moieties described herein. Derivatives include modifications to drugs identified herein which may improve or not significantly reduce a particular drug's desired therapeutic activity. [00105]The term "second agent" includes any moiety, proteinaceous or non- proteinaceous, which is attached to a first agent through a linking group using methods described herein. In some embodiments, the second agent is a proteinaceous molecule such as, for example, a polypeptide, an antibody, a fusion protein or a hormone. Examples of proteinaceous second agents include, but are not limited to, interleukins 1
through 18, including mutants and analogues; interferons α, β, and γ; luteinizing
hormone releasing hormone (LHRH) and analogues, gonadatropin releasing hormone
(GnRH), transforming growth factor-β (TGF-β); fibroblast growth factor (FGF); tumor
necrosis factor-α & β (TNF-α & β); nerve growth factor (NGF); growth hormone
releasing factor (GHRF); epidermal growth factor (EGF); fibroblast growth factor homologous factor (FGFHF); hepatocyte growth factor (HGF); insulin growth factor (IGF); platelet-derived growth factor (PDGF); invasion inhibiting factor-2 (IIF-2); bone
morphogenetic proteins 1-7 (BMP 1-7); somatostatin; thymosin-α -1; γ-globulin;
superoxide dismutase (SOD); and complement factors, hormones such as, for example, insulin, interferons or cytokines and other bioactive peptidic compounds, such as hGH, tPA, calcitonin, ANF, EPO, insulin; antibodies (e.g., human anti-TAC antibody), recombinant beta-glucan; bovine immunoglobulin; bovine superoxide dismutase; recombinant hirudin (r-Hir), HIV-I immunogen; recombinant human growth hormone (r-hGH); recombinant human hemoglobin (r-Hb); recombinant human mecasermin (r- IGF-I); recombinant interferon beta-la; lenograstim (G-CSF); olanzapine; recombinant thyroid stimulating hormone (r-TSH); and topotecan. Without wishing to be bound by theory, it is contemplated that any protein can be labeled using methods of the invention by simply fusing the protein to an alpha-helical oligopeptide moiety, as described herein. [00106]Generally, the second agent is linked to an alpha-helical oligopeptide moiety, as described above. When the second agent is a proteinaceous molecule, it may be linked through its N-terminus to an alpha-helical oligopeptide moiety (e.g., an alpha-helical oligopeptide moiety having an amino acid sequence set forth in SEQ 3D NO: 1 or 2 or 40 or 41 or a variant thereof). Furthermore, when the second agent is proteinaceous, the second agent may be linked to one or more alpha-helical oligopeptide moieties having an amino acid sequence set forth in SEQ H) NO:2 or SEQ ID NO:40 or 41 or a variant thereof at its N-terminus, the C-terminus or anywhere in between the N-terminus and the C-terminus.
[00107]In other embodiments, the second agent may be a non-proteinaceous moiety such as, for example, a nucleic acid molecule or a derivative thereof, a lipid molecule or a derivative thereof, or a carbohydrate or a derivative thereof. A second agent may also be a small molecule moiety, as described herein. Other examples of second agents include, for example, an affinity probe, a spectroscopic probe, a radioactive probe, a radical generating molecule, a singlet oxygen generating molecule, a polymer, a hapten, a chelating agent or a receptor binding molecule, which may be linked to at least one alpha-helical oligopeptide moiety, such as, for example, an alpha helical moiety having an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40 or SEQ ID NO:41, or a variant thereof. In yet other embodiments, the second agent includes at least two portions, a proteinaceous portion and a non-proteinaceous portions. The second agents may further comprise one or more epitope tags such as, for example, a flag tag, a GST tag, a His tag, an HA-tag. Such tags are especially useful for the purification of the second agent linked to a first agent, using the methods described herein. [00108]The term "alpha-helical oligopeptide moiety," refers to an oligopeptide or variant thereof which is capable of forming an alpha helical structure either when linked to another peptide (for example, in case of the amino acid sequence set forth in SEQ ID NO:1, or a variant thereof); or is capable of forming an alpha helical structure independent of the second agent (for example, the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40 or SEQ ID NO:41 or a variant thereof). The term "alpha- helical oligopeptide moiety" includes peptides which are capable of forming an alpha- helical structure in solution such as, for example, peptides including an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40, or a variant thereof, and those peptides which are capable of forming an alpha-helical structure upon binding to an enzyme having phosphopantetheinyl transferase activity such as, for example, the peptide having an amino acid sequence set forth in SEQ ID NO:41, or a variant thereof. [00109] Generally, the alpha-helical oligopeptide moiety is linked to a second agent and is capable of accepting a first agent-linking group conjugate via a phosphopantetheinylation reaction. In some embodiments, the alpha-helical oligopeptide moiety includes the amino acid sequence DSL and at least two other alpha- helix favoring amino acids. In yet other embodiments, the alpha-helical oligopeptide moiety includes the amino acid sequence DSL and at least three other alpha helix favoring amino acids. In some other embodiments described herein, an alpha-helical oligopeptide moiety is capable of forming an alpha-helical structure independent of the second agent to which it is linked. For example, in some embodiments, the alpha-helical oligopeptide moiety includes the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40 or SEQ ID NO:41, or a variant thereof, which can form an alpha-helical structure independently of the second agent, to which it is linked, either in solution or upon binding to an enzyme having phosphopantetheinyl transferase activity. Alpha- helical oligopeptide moieties may either include D-amino acids, L-amino acids, or a combination thereof. Also, encompassed by this disclosure are nucleic acid molecules encoding the alpha-helical oligopeptide moieties described herein. [00110] Variants of alpha helical oligopeptide moieties include peptides which include one or more amino acid mimetics and are capable of accepting a first agent-linking group conjugate in a phosphopantetheinylation reaption. An "amino acid mimetic" refers to a moiety, other than a naturally occurring amino acid, that conformationally and functionally serves as a substitute for a particular amino acid in a peptide compound without adversely interfering with the function of the peptide. [00111] "Alpha-helix favoring amino acids," are those amino acids which favor formation of an alpha helical structure when present in a peptide. Exemplary alpha helix favoring amino acids include, for example, Ala, GIu, Leu and Met. Alpha-helix favoring amino acids, as used herein, specifically exclude those amino acids which either prevent or hinder the formation of an alpha helical structure when included in a peptide. Examples of such amino acids include Pro, GIy, Tyr and Ser. [00112]The term "linking group," includes moieties which can be transferred from a donor molecule {e.g., coenzyme A) to an alpha-helical oligopeptide moiety via a phosphopantetheinylation reaction. As used herein, linking groups are typically linked to a first agent, which along with the linking group is transferred to an alpha-helical oligopeptide moiety. For example, the linking group may be linked to I5 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 first agents. Linking groups and first agents are selected such that they are capable of being transferred to the alpha-helical oligopeptide moieties encompassed by this disclosure, through a phosphopantetheinylation reaction.
[00113]In one embodiment, the linking group is phosphopantetheine or a derivative thereof, hi a further embodiment, the linking group (shown linked to a first agent M) is of the formula:
Figure imgf000047_0001
wherein each R is M, optionally substituted alkyl, halogen, alkoxy, hydroxy, or hydrogen independently selected for each occurence;
U1 is oxygen, sulfur or N-M;
U2 is O-M, S-M, CR2-M, or NRM; and each M is an independently selected first agent for each occurrence. [00114] hi a further embodiment, the linking group (shown linked to a first agent M) is of the formula:
Figure imgf000048_0001
wherein each R is M, optionally substituted alkyl (e.g., CM3, etc.), halogen, alkoxy, hydroxy, or hydrogen independently selected for each occurence;
U1 is oxygen, sulfur or N-M;
U2 is O-M, S-M, CR2-M, or NRM; and each M is an independently selected first agent for each occurrence. [00115]In another further embodiment, the linking group linked to a first agent
(M) is of the formula:
Figure imgf000048_0002
wherein each R is M, optionally substituted alkyl (e.g., CM3, etc.), halogen, alkoxy, hydroxy, or hydrogen independently selected for each occurence;
U1 and U3 are each independently oxygen, sulfur or N-R;
U2 is oxygen, sulfur, CR2, or N-M;
U4 is NR2, CR3, S-R or O-R; and each M is an independently selected first agent for each occurrence. [00116]Examples of linking groups linked to first agents {e.g., Mq-L) include moieties of the formula (V):
Figure imgf000049_0001
wherein each M is an independently selected first agent for each occurrence. [001 17]In other embodiments, Mq-L is of the formula (VI):
Figure imgf000049_0002
wherein each M is an independently selected first agent for each occurrence. , [00118]The term "leaving group," as used herein, includes groups that when linked to the linking group and first agent are capable of departing in a phosphopantetheinylation reaction, resulting in the transfer of the linking group and first agent to the alpha-helical oligopeptide moiety. Examples of leaving groups include phosphorylated nucleotides such as 3', 5'-ADP.
[00119] Also encompassed by this invention are pharmaceutical compositions including one or more molecules produced by the methods described herein. In some embodiments, pharmaceutical compositions further include a pharmaceutically acceptable carrier. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration into a subject. [00120] Without wishing to be bound by theory, it is contemplated that Sfp and Acps catalyzed posttranslational modification of molecules linked to an alpha-helical oligopeptide moiety, as described herein, can have several applications both in vitro and in vivo.
[00121]Methods of the invention can be used, for example, for labeling two proteins with two separate compounds. For example, in some embodiments of the present invention, two proteins present in a cell lysate can be labeled using the methods of the invention using a Sfp catalyzed reaction for labeling one protein and an Acps catalyzed reaction for labeling a second protein. Accordingly, one protein can be linked with one or more alpha-helical oligopeptide moieties including an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40, and the second protein can be linked with an alpha- helical oligopeptide moiety which includes the amino acid sequence set forth in SEQ ID NO:41. The cell lysate including both proteins (e.g., cell lysate derived from cells expressing both proteins linked to the respective alpha-helical oligopeptide moieties) can be contacted first with the Sfp enzyme or a fragment thereof, having phosphopantetheinyl transferase activity in the presence of a first compound, thereby labeling the protein linked to the alpha-helical oligopeptide moiety including the amino acid sequence set forth in either SEQ ID NO:2 or 40. Subsequently, the cell lysate can be contacted with the Acps enzyme or a fragment thereof, having phosphopantetheinyl transferase activity in the presence of a second compound, thereby labeling the protein linked to the alpha-helical oligopeptide moiety including the amino acid sequence set forth in SEQ ID NO:41. Alternatively, an Acps catalyzed reaction can be carried out before the Sfp catalyzed reaction.
[00122] Such a method can also be used for labeling one or more proteins on the surface of live cells. [00123]For example, in some embodiments, in some embodiments, a method of delivering a second agent such as, for example, a protein, to a desired location in a subject is described. A method of delivering a second agent to a desired location within a subject, includes for example, administering to a subject a molecule of formula (I), where the first agent is capable of delivering the second agent to the desired location within the subject, where the molecule of formula (I) is:
Mq-L-Y-Z (I) where:
M is a first agent; q is an integer greater than 0;
L is a linking group;
Y is an alpha-helical oligopeptide moiety; and
Z is a second agent.
[00124]In some embodiments, molecules made using methods described herein can be used for disease detection and/or diagnosis. For example, in some embodiments, a second agent is molecule which recognizes a tumor specific antigen (e.g., an antibody which binds a tumor specific antigen). Thus, an antibody or a fragment thereof which specifically binds a tumor specific antigen, for example, can be linked to a first agent (e.g., a suitable tag for fluorescent imaging) using methods described herein and administered to a subject, where detection of the fluorescent first agent is indicative of the presence of the tumor in the subject.
[00125]ln other embodiments, molecules made using methods described herein can be used for simultaneous administration of two or more agents to a subject, for example, for treatment of a disorder or a disease, where combination of the two or more agents is effective for treatment. For example, in some embodiments, a first agent can be a hormone (e.g., insulin) used for the treatment of diabetes, and a second agent can be a drug which is commonly used in conjunction with insulin for the treatment of diabetes. Therefore, insulin conjugated with a second agent can be generated using methods described herein and used in the treatment and/or management of diabetes. [00126]Tbis invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference.
Examples Materials and Methods
I. Chemical synthesis:
[00127]Synthesis of biotin-CoA has been previously reported (Yin et al., J. Am. Chem. Soc, 126:7754-55, 2004). Fluorescein - CoA , tetramethylrhodamine - CoA and Texas Red - CoA were synthesized using a similar procedure. Briefly, to a solution of
coenzyme A trilithium salt (SIGMA) (16.5 mg, 22 μmol) in 2 mL sodium phosphate 100
mM, pH 7.0, fluorescein-5-maleimide (MOLECULAR PROBES, F150) (8.5 mg, 20 μmol) in 0.5 mL DMSO, or tetramethykhodamine-5-maleimide (MOLECULAR PROBES, T6027) (5 mg, 10 μmol) in 0.5 mL DMSO, or Texas red C2 maleimide (MOLECULAR PROBES, T6008) (5 mg, 6.9 μmol) in 0.5 mL DMSO, were added. The reaction mixtures were stirred at room temperature for about one hour in dark followed by purification with preparative HPLC on a reverse-phase Cl 8 column with a gradient of 0-50% acetonitrile in 0.1% TFA/water for 30 minutes. The purified compound was lyophilized and the identity was confirmed by MALDI-TOF (negative mode): [M-H]", fluorescein - CoA, calculated 1193.2, observed 1192.8; tetramethylrhodamine - CoA, calculated 1248.3, observed 1247.8; and Texas Red - CoA, calculated 1509.3, observed 1509.2. Peptides were synthesized by the Biopolymer Laboratory of the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School. AU peptides were purified by preparative HPLC and the mass of the peptides were confirmed by MALDI-TOF before kinetic experiments. [00128]Biotin~SS-CoA was synthesized with the following procedure. To a solution of biotin HPDP (PIERCE) (10.8 mg, 0.020 mmol) in 500 μL DMSO, coenzyme A lithium salt (SIGMA) (16.5 mg, 0.022 mmol) in 2 ml sodium phosphate, 100 mM, pH 7.0, was added and the reaction mixture was stirred at room temperature for one hour. The reaction mixture was then purified by preparative HPLC on a reversed-phase Cl 8 column with a gradient of 0-60% acetonitrile in 0.1% TF A/water over 35 minutes. The purified compound was lyophilized and its identity was confirmed by LC-MS (negative mode) [M-H"]: 1194.72, calculated for C40H67NnO19P3S3 ": 1194.31. Synthesis of Alexa 488-CoA and Texas red-CoA was previously reported (Yin et ah, Proc. Natl. Acad. Sci. USA 102:15815-15820 (2005)).
II. Construction of the phage displayed peptide library.
[00129JDNA primers Junl89 (5'- CAG GCG GCC GAG CTC GGT GGA GAT TCT MTT NNKNNK NNK NNK NNKNNKNNKNNK GGT GGC CAG GCC GGC CAG, M=A or C, N-A or G or C or T, K = G or T) (SEQ ID NO:42) and Junl91 (5' - CGC TTA CAA TTT CCC AGA TCT GCG) (SEQ ID NO:43) were used to amplify a 600 base pair fragment from the phagemid vector pcomb3H-7G12 (Weber et al., Structure 8:407-418 (2000)) and cloned into the same vector between the Sad and Nhel restriction sites. Subsequent to the ligation reaction, the phagemid DNA was transformed into XLl -Blue competent cells (STRAT AGENE) by electroporation and the cells were plated on LB agarose plates supplemented with 2% (w/v) glucose and 100 μg/mL ampicillin. After incubation at 37°C overnight, colonies on the plates were picked and the phagemid DNA was extracted by a Qiagen Plasmid Maxi kit (QIAGEN).
III. Cloning
[00130] Phagemids including various lengths of the ybbR gene were isolated from the selected phage clones by a DNA miniprep ldt (QIAGEN). The B. suhtilis genomic DNA fragments were sequenced using primers Junl3 (5'- ACT TTA TGC TTC CGG CTC GTA TGT) (SEQ ID NO:7) and Junl4 (5'- AAT CAA AAT CAC CGG AAC CAG AGC) (SEQ ID NO: 8) and the ybbR gene fragment (from clone JY529) was subcloned into the ρET-21b plasmid (NOVAGEN) using the EcoRI and HindIII restriction sites for enabling the expression of JY529 as a fusion protein with a C-terminal 6χHistidine tag (JY529-His). Site directed mutagenesis of Serine at position 274 to Ala (Ser274Ala) in JY529 was accomplished using the Quickchange site directed mutagenesis kit (STRATAGENE) using the primers Junl69 (5'- CAG GAT GTG TTG GAT GCT CTA GAA TTT ATT GCT AGT AAG C) (SEQ ID NO:9) and Junl70 (5'- G CTT ACT AGC AAT AAA TTC TAG AGC ATC CAA CAC ATC CTG) (SEQ ID NO: 10). [00131 ]For the construction of a ybbR-EGFP fusion protein, primers Jun 167 (5 ' - GAA TCC AGC CCC CAT ATG GTG TTG GAT TCT CTT GAA TTT ATT GCT AGT AAA CTG GCG AAG CTT GTG AGC AAG GGC GAG G) (SEQ ID NO: 11) and Jun57 (5'- AGA GTC GCG GCC CTC GAG CTT GTA CAG CTC GTC C) (SEQ ID NO: 12) were used to amplify the EGFP gene from the plasmid pEGFP (CLONETECH, BD BIOSCIENCES) by PCR, which resulted in linking the DNA sequence encoding the ybbR12 peptide 5' to the EGFP gene and introduced restriction sites Ndel and Xhol at the 5' and 3'-ends of the ybbR12-EGFP PCR fragment. Primer Junl67 also inserted a HindIII site in between the ybbR12 coding sequence and the EGFP gene. The PCR fragment was subsequently cloned into pET22b vector (NOVAGEN) between the Ndel and Xhol sites resulting in the pET22b-ybbR12-EGFP construct for the expression of EGFP with ybbR12 fused to the N-terminus and a 6xHistidine tag at the C-terminus. [00132]GST and MBP genes were amplified using primer pairs Jun58 (5'- GAA ACA GTA TTC AAG CTT CCT ATA CTA GGT TAT TGG) (SEQ ID NO:13) / Jun60 (5'- GGA TCC ACG CGG CTC GAG ATC CGA TTT TGG) (SEQ ID NO: 14 ) and Junόl (5'- GGA CCA TAG CAT AAG CTT ATC GAA GAA GGT AAA CTG G) (SEQ ID NO:15) / Jun63 (5'- CAG GTC GAC TCT CTC GAG TCC GAA TTC TGA AAT CC) (SEQ ID NO: 16), respectively, which introduced Hindiπ and Xhol sites at the 5' and 3'- ends, respectively, of the PCR fragments. The amplified GST and MBP genes were subsequently cloned into ρET22b-ybbR12-EGFP between the HindIII and Xhol sites, resulting in the constructs ρET22b-ybbR12-GST and pET22b-ybbR12-MBP for the expression of N-terminal ybbR tagged ybbR-GST and ybbR12-GST fusions, respectively. Similarly, primers Junl69 (5'- GAA TCC AGC CCC CAT ATG GAT TCT CTT GAA TTT ATT GCT AGT AAA CTG GCG AAG CTT GTG AGC AAG GGC GAG G) (SEQ ID NO: 17) and Jun57 were used to PCR amplify EGFP genes for the construction of expression vector pET-ybbR13-EGFP which expresses the N- terminal ybbR13-EGFP fusion. Primers Junl68 (5'- GAA TCC AGC CCC CAT ATG GAT TCT CTT GAA TTT ATT AAG CTT GTG AGC AAG GGC GAG G) (SEQ ID NO: 18) and Jun57 were used to construct the expression vectors for the expression of N- terminal ybbR peptide with the sequence DSLEFI (SEQ ID NO: 19) fused to EGFP. [00133]For the construction of ybbR tags fused to the C-terminus of EGFP, primers Junl73 (5'- TTG TTA GCA GCC GGA TCC TCA CGC CAG TTT ACT AGC AAT AAA TTC AAG AGA ATC CAA CAC CTC GAG CTT GTA CAG CTC GTC CAT GCC G) (SEQ ID NO:20) and Junl72 (5'- CCC CGG GTA CCG CAT ATG AAG CTT GTG AGC AAG GGC GAG G) (SEQ ID NO: 21) were used to PCR amplify the EGFP gene with the coding sequence for ybbR12 linked to the 3 'end of the EGFP gene. The amplified PCR fragment further included the Ndel and BamHI sites at the 5' and 3 '-ends respectively for the cloning of the fragment into pET14b (NOVAGEN), resulting in the plasmid ρET14b-EGFP-ybbR12. EGFP including ybbR12 fused to the C-terminal and a 6χHistidine tag at the N-terminal was subsequently expressed. Primer Junl73 also introduced an Xhol site at the end of EGFP gene immediately before the ybbR12 coding sequence.
[00134]PCR amplified gene fragments of GST and MBP using primers Junl81 (5'- GAA ACA GTA TTC CAT ATG CCT ATA CTA GGT TAT TGG) (SEQ ID NO:22) / Jun60, and Junl82 (5'- GGA CCA TAG CAT CAT ATG ATC GAA GAA GGT AAA CTG G) (SEQ ID NO:23) and Jun63 respectively, included Ndel and Xhol at the 5' and 3' end respectively and were subsequently cloned into the pET14b-EGFP-.ybbR12 vector between those two restriction sites for the expression of GST and MBP fusions with ybbR12 peptide tag fused to the C-terminus.
[00135]For the construction of ybbR12 peptide inserted in the middle of a EGFP gene, primer pairs Junl 86 (5'- GGC GGA GGT ACC GTG TTG GAT TCT CTT GAA TTT ATT GCT AGT AAG CTT GCG GGT GGA GGC AAC AGC CAC AAC GTC TAT ATC ATG) (SEQ ID NO:24)/ Jun57, and Junl 87 (5'- CAA CAC GGT ACC TCC GCC GTT GTA CTC CAG CTT G) (SEQ ID NO:25) / Junl88 (5'- GAA TCC AGC CCC CAT ATG GTG AGC AAG GGC GAG G) (SEQ ID NO:26) were used for PCR amplifying two fragments of the EGFP gene and upon overlap assembly of these two fragments by PCR with primers Junl88 and Jun57, coding sequence of the ybbR12 peptide with flanking GIy residues was inserted in the place of Tyrl45 in the EGFP gene and the assembled gene fragment was cloned into the pET22b vector between the Ndel and Xhol sites. IV. Expression of Sfp and Acps.
[00136]The Acps gene was amplified from pPDJ (Yin et ah, J. Am. Chem. Soc, 126:3006-3007(2004)) with primers 5'-TCT GGT CAT ATG GCA ATA TTA GGT TTA GGC ACG G (SEQ ID NO:44) and 5'- TCA AGT CTC GAG TTA ACT TTC AAT AAT TAC CGT GGC A (SEQ ID NO:45). The resulting PCR product was digested with Ndel and Xhol and cloned into corresponding restriction sites of the pET28b vector (NOVAGEN). ACPS protein was overexpressed in BL21 DE3 star cells (DSrVITROGEN) at 3O0C for 6 hours. The N-terminal His-tagged recombinant ACPS protein was purified with standard Ni-NTA purification procedures and dialyzed into buffer containing 100 mM Bis-Tris propane (pH 6.0), 500 niM NaCl and 10% glycerol. Expression of Sfp was carried out, as previously reported (Yin et ah, Nature Protocols 1:280-285 (2006)).
V. Preparation of the phage particles.
[00137]Phage particles were produced following procedures as previously described (Weber et ah, Structure 8:407-418 (2000)). Briefly, E. coli XLl-Blue cells were transformed with pComb vectors, and shaken at 37°C in 2X yeast/tryptone broth and 100 μg/ml ampicillin. At an OD600 of 0.5, helper phage VCSM13 (STRATAGENE) was added to a final concentration of 1.5 X 10s cfu/ml, and incubated at 37°C for 1 hour without shaking. The cells were pelleted and resuspended in 2X YT, 100 μM isopropyl- D-thiogalactoside (IPTG), 100 μg/ml ampicillin, and 50 μg/ml kanamycin, and shaken for 14 h at room temperature. The following day, cells were pelleted and phage particles in the supernatant were precipitated using polyethylene glycol, followed by resuspension in TBS (25 mM Tris-HCl, pH 7.4/140 mM NaCl/2.5 mM KCl). Phage titrations were performed with E. coli XLl -blue using standard procedures. VI. Phage selection.
[00138]Phage-displayed peptides were first labeled with biotin by Sfp or Acps using biotin-SS-CoA as the substrate. For the first round of selection, the labeling reactions were carried out with 1012 phage particles in 1 mL 10 mM MgCl2 and 50 niM HEPES pH 7.5 with 5 μM biotin-SS-CoA, 1 μM Sfp or 5 μM Acps and incubated at 30°C for 30 minutes. For the subsequent rounds of selections, the number of the input phage particles, concentration of enzymes and biotin-SS-CoA and the reaction time were decreased step by step and eventually for the fifth round of selection, only 1010 phage particles were incubated with 0.08 μM enzyme and 1 μM biotin-SS-CoA for 7 minutes at 30°C. Control reactions were also run in parallel without the addition of enzymes or biotin-SS-CoA.
[00139] After the labeling reaction, the reaction mixtures were added to 250 μL of 20% (w/v) polyethylene glycol 8000 with 2.5 M NaCl followed by 10 minute incubation on ice. The phage particles in the reaction mixture were then precipitated by centrifugation at 40C at a speed of 13,000 revolutions per minute (rpm). The phage pellet was subsequently resuspended in 1 mL TBS supplemented with 1% (w/v) BSA and distributed in 100 μL aliquots to the wells of streptavidin coated 96 well plates (PIERCE). The plates were allowed to incubate at room temperature for one hour before the supernatant was discarded and each well was washed 30 times with 0.05% (v/v) Tween 20, 0.05% (v/v) Triton X-100 in TBS and 30 times with TBS, each time with 200 μL of solution. After washing, phages bound to the streptavidin surface were eluted by adding 100 μL 20 mM dithiothreitol in TBS to each well to induce the cleavage of the disulfide bond that links the biotin group with Ppant. Eluted phage particles were combined, added to 10 mL of log phase XLl -Blue cells and shaken at 370C for one hour to infect the cells. The cells were then plated on LB agarose plates supplemented with 2% (w/v) glucose and 100 μg/mL ampicillin. After incubation at 37°C for overnight, colonies on the plates were scratched and the phagemid DNA was extracted by a Qiagen Plasmid Maxi kit. The phagemid DNA was then used for the next round of phage production and selection. Also phage particles eluted from the wells loaded with either the selection and the control reactions were titered in order to count the number of phage particles selected by each round. After the fifth round of selection, phage clones were sequenced using the primer Junl3 (5'- ACT TTA TGC TTC CGG CTC GTA TGT) (SEQ ID NO:7).
VII. Phage ELISA.
[00140]Phages (1010 particles) displaying S or A series of peptides were labeled with biotin by Sfp or Acps under the same conditions as the labeling reaction for phage selection with 0.5 μM of enzyme and 1 μM of biotin-CoA. Control reactions were also run in parallel with the exclusion of biotin-CoA or enzyme. After incubation at 30°C for 30 minutes, 20 μL of the reaction mixtures were added to 80 μL 1% BSA in TBS in the wells of a 96 well plate coated with streptavidin. Cross-plate series dilution of the reaction mixtures was carried out by transferring 20 μL of the reaction mixture in 1% BSA to the wells in the column using a multichannel pipetor. The streptavidin plate was then incubated at room temperature for 60 minutes and washed five times with 0.05% (v/v) Tween 20, 0.05% (v/v) Triton X-100 in TBS, followed by washing five times with TBS, each time with 200 μL of solution. 100 μL 1/5000 diluted anti-M13 HRP conjugate (GE HEALTHCARE) in 1% BSA in TBS was added to each well and incubated at room temperature for another hour. The plate was again washed five times with 0.05% (v/v) Tween 20, 0.05% (v/v) Triton X-100 in TBS and five times with TBS. The plate was subsequently developed by adding 100 μL TMB peroxidase substrate and hydrogen peroxide mixture (PIERCE) to each well and incubated at room temperature for five minutes before being photographed.
Viπ. Fourier Transform Mass Spectrometric Analysis
[00141]In order to investigate Ppant modification of JY529-His by Sfp, JY529-His (1.4 mM) was combined with Sfp (4 μM) and CoA (1 mM) to the parenthetically indicated final concentrations in 100 mM Tris-HCl, pH 8.0, 10 mM MgCl2 and 1 mM TCEP, and incubated at 3O0C for 4 hours to allow for phosphopantetheinylation. The mixture was directly injected onto a 4.6 x 150 mm Jupiter C4 reversed phase column and eluted in a linear gradient of 15 to 55% CH3CN over 40 minutes at a flow rate of 1 mL min"1. The absorbance was monitored at 220 nm and two peaks eluting at 26 and 29 minutes were collected. The fractions were lyophilized and re-suspended in 200 μL of 50:50 H2OiCH3CN with 0.1% formic acid and directly infused into a custom built quadrupole Fourier-Transform mass spectrometer (Q-FTMS) via a Nanomate nanoelectrospray robot. After the ions were passed through a resistively heated metal capillary/ they were externally accumulated in an octupole for 500 ms (Senko et al., J. Am. Mass. Spectrom, 8:970-976, 1997). The ions were subsequently shuttled to the analyzer cell through a quadrupole that can act as a simple ion guide or a filter for selected m/z windows. The data were fit using a least squares algorithm THRASH (Horn et al., J. Am. Soc. Mass Spectrom, 11 : 320-332, 2000) and the monoisotopic and most abundant isotopic peaks were determined. Tandem mass spectrometry was accomplished by first using the quadrupole to mass select specific charge states prior to subjecting the ions to octupole collisionally activated dissociation (OCAD), infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD). IX. Peptide Labeling Reaction
[00142]In order to determine whether a peptide (e.g., an alpha-helical oligopeptide moiety) was the substrate of Sfp catalyzed biotin - CoA modification, to a 100 μL solution of 100 μM peptide in 10 mM MgCl2 and 50 mM HEPES, pH 7.5, Sfp and biotin - CoA were added to a final concentration of 200 μM of biotin - CoA and 1 μM Sfp,
and incubated at 37°C for 60 minutes. Control reactions were also run in parallel with
either Sfp or biotin - CoA excluded from the reaction. Reactions were then quenched by adding 30 μL 4% trifluoroacetic acid (TFA) and analyzed using analytical HPLC with a reverse phase C18 column using a gradient of 0-60% CH3CN in 0.1% TFAZH2O over 30 minutes and monitored at 220 nm. Peptide labeling reactions were also carried out at various pHs ranging from 5.0 to 8.5 with various buffering reagents (pH 5.0, sodium acetate 50 mM; pH 6.0, MES, 50 mM; pH 7.0 HEPES 50 mM; pH-8.0, HEPES 50 mM; pH 8.5 Tris-HCl 50 mM) to test the effect of pH on the rate of Sfp catalyzed peptide labeling.
[00143]For the determination of kinetic parameters of Sfp catalyzed peptide labeling at saturating concentration of biotin - CoA or fluorescein - CoA, Sfp was added to a final concentration of 1 μM in 10 mM MgCl2 and 50 mM HEPES, pH 7.5 buffer, with varying concentration of the peptide ranging from 2 μM to 500 μM, while holding the concentration of the biotin - CoA or fluorescein - CoA conjugate constant at 150 μM. For the determination of kinetic parameters at saturating concentration of the peptide, the peptide concentration was kept constant at 500 μM and the biotin - CoA or fluorescein - CoA concentration was varied from 2 μM to 200 μM. The reaction was
allowed to proceed at 37°C for 5 minutes, quenched and analyzed by HPLC as described
above. HPLC peak areas were integrated, and the product concentration was calculated as a percent of the total peak area. Initial velocity data were fit to the Michaelis-Menten equation by using the program KaleidaGraph.
[00144]The kinetic parameters for the Sfp catalyzed PCP labeling was carried out in the same buffer (10 mM MgCl2 and 50 mM HEPES, pH 7.5) in presence of 0.1 μM of Sfp by either varying the concentration of PCP from 0.5 μM to 50 μM at a constant biotin - CoA concentration of 150 μM or varying the biotin - CoA concentration from 2 μM to 200 μM at a constant PCP concentration of 20 μM. The reaction was allowed to
proceed at 37°C for 5 minutes before quenching by the addition of 30 μL 4% TFA to 100 μL reaction mixture. The reaction was then analyzed by analytical HPLC with a reverse phase C18 column using a gradient of 30-50% CH3CN in 0.1% TFAZH2O over 30 minutes and monitored at 280 nm.
X. Circular Dichroism Spectroscopy
[00145]Peptides (e.g., alpha helical oligopeptide moieties) were suspended in solution with 30% 2,2,2-trifluoroethanol (TFE) and 5mM potassium phosphate at pH 7.5. All the peptides had a concentration of 0.2 mg/ml, except for the TycC3 peptide, and were prepared to a concentration of 0.1 mg/ml for solubility purposes. Circular dichroism ellipticity was recorded using an AVIV 60DS instrument. Spectra from 260 nm to 190 nm were scanned at a step of 1 nm at 25 0C in a 0.1 cm cuvette, with 3 repeats and an averaging time of 3 seconds. The data were finally expressed as the mean residue molar ellipticity,
Figure imgf000062_0001
The helix contents of peptides were estimated by Greenfield and Fasman equation (Plenum Press, New York, 1996).
XL Labeling Second Agents with biotin
[00146]Proteins fused to a ybbR peptide and including a His-tag were expressed and purified as όxHistidine fusion proteins using Ni-NTA affinity resin following standard protocols. Sfp catalyzed biotin CoA labeling of the purified ybbR tagged proteins was performed following the procedure described in (Yin et al. J. Am. Chem. Soc, 126:7754-55, 2004). In a total volume of 100 μL, 0.5 μM Sfp, 5 μM biotin - CoA, 10
mM MgCl2 in 50 mM HEPES3 pH 7.5, were incubated at 370C with 10 μM ybbR tagged
protein for 15 minutes. To detect biotin labeling, cell lysates containing 2 μg protein were loaded on to a 4-15% SDS-PAGE gel (BIORAD). Following electrophoresis, the protein bands were electroblotted onto a piece of PVDF membrane (BIORAD). The membrane was then blocked with 3% BSA in Tris-buffered saline (TBS) for 2 hours followed by incubation with 0.1 μg/ml streptavidin - HRP conjugate (PIERCE) in 1% BSA for 1 hour. The membrane was subsequently washed about five times with 0.05% Tween 20 and 0.05% Triton X-100 in TBS followed by five washes in TBS alone. Streptavidin-HRP binding was detected using an ECL luminescence detection kit (AMERSHAM PHARMACIA). -
[00147]For the labeling reaction with cell lysates, 1 ml of a culturέ of E. coή
expressing ybbR tagged proteins was grown overnight at 30°C following PTG
induction. The cells were pelleted by centrifugation, re-suspended in 1 mL HEPES 50 mM, pH 7.5 buffer and sonicated for 1 minute to prepare the cell lysates. 10 μL cell lysates was then added to 90 μL 10 mM MgCl2, 50 mM HEPES, pH 7.5 buffer, with 0.5
μM Sfp and 5 μM biotin - CoA and incubated at 37°C for 15 minutes. The labeling
reaction was then analyzed by PAGE electrophoresis and western blot. [00148]The procedures for phage displayed ybbR protein labeling by biotin - CoA and subsequent phage ELISA was previously reported. (Yin et al. J. Am. Chem. Soc. 126:13570-13571, 2004).
XII. Labeling Second Agents with Fluorophores
[00149]Purifϊed EGFP including a ybbR tag inserted at position 145 was labeled with tetramethylrhodamine and Texas Red by Sfp using tetramethylrhodamine - CoA and TexasRed - CoA, and following the same procedure as labeling using biotin - CoA. The labeling reaction mixture was then loaded onto an Econo-Pac IOODG desalting column (BIORAD) to remove the un-reacted fluorophore - CoA conjugates in the solution. Fractions corresponding to EGFP eluted from column were collected and measured for UV absorbance.
XIII. Kinetics of the Peptide Labeling Reaction Catalyzed by Sfb and Acps. [00150]Peptides were synthesized at the HMS BCMP Biopolymers Laboratory and purified by high performance liquid chromatography (FJPLC). The kinetics of PPTase- catalyzed peptide labeling reaction by biotin-CoA was measured following the procedures previously reported (Yin et ah, Proc. Natl. Acad. Sci. USA, 102:15815- 15820 (2005)). For the labeling of S series peptides with Sfp, 0.66 μM Sfp was used with a reaction time of 5 or 10 minutes at 370C. For the labeling of A series peptides with Acps, 1 μM Acps was used with a reaction time of 7.5 or 15 minutes. For the labeling of S series peptides with Acps or A series peptides with Sfp, 5 μM Sfp or Acps were used with a reaction time of 60 or 120 minutes at 37°C.
XTV. Construction of S and A Peptide Tagged Protein Fusions. [00151]For the construction of N-terminal Sl- and Al-tagged EGFP fusions, primers Jun219 (5'- GAA TCC AGC CCC CAT ATG GGA GAT TCT CTT TCG TGG CTG GTT AGG TGT TTG AAT GGT AAG CTT GTG AGC AAG GGC GAG G) (SEQ ID NO:46) and Jun220 (5'- GAA TCC AGC CCC CAT ATG GGA GAT TCT CTT GAT ATG TTG GAG TGG TCT TTG ATG GGT AAG CTT GTG AGC AAG GGC GAG G) (SEQ ID NO:47) were used to pair with Jun57 (5'- AGA GTC GCG GCC CTC GAG CTT GTA CAG CTC GTC C) (SEQ ID NO:12) in order to amplify Sl-EGFP and Al- EGFP gene fusions respectively from the plasmid pET22b-ybbR12-EGFP (Yin et ah, Proc. Natl. Acad. Sci. USA, 102:15815-15820 (2005)). The PCR fragments were then cloned into the pET22b vector (NOVAGEN) between the restriction sites of Ndel and Xhol to give the plasmids of pET22b-Sl-EGFP and pET22b-Al-EGFP for the expression of N-terminal Sl and Al tagged and C-terminal 6><His tagged EGFP proteins. Jun233 (5'- TCC AGC CCC CAT ATG GGA GAT TCT CTT TCG TGG CTG CTT AGG TGT TTG AAT GG) (SEQ 3D NO:48) and Jun234 (5'- TCC AGC CCC CAT ATG GGA GAT TCT CTT TCG TGG CTG CTT AGG CTT TTG AAT GGT AAG C) (SEQ ID NO:49) were then used to pair with Juii57 in order to amplify 5'- S2 and S6 fused EGFP genes from vector pET22b-Sl-EGFP. The PCR fragments were cloned into pET22b vector to give plasmids ρET22b-S2-EGFP and pET22b-S6~EGFP for the expression of N-terminal S2 and S6 tagged and C-terminal 6x His tagged EGFP proteins. For the construction of C-terminal Sl and Al tagged EGFP fusions, Jun221 (5'- TTG TTA GCA GCC GGA TCC TCA ATT CAA ACA CCT AAC CAG CCA CGA AAG AGA ATC TCC GCC CTC GAG CTT GTA CAG C) (SEQ ID NO:50) and Juii222 (5'- TTG TTA GCA GCC GGA TCC TCA CAT CAA AGA CCA CTC CAA CAT ATC AAG AGA ATC TCC GCC CTC GAG CTT GTA CAG C) (SEQ ID NO:51) were paired with Junl76 (5'- TAA TAC GAC TCA CTA TAG GG) (SEQ ID NO:52) in order to amplify 3'- Sl and Al fused EGFP genes from vector pET14b-EGFP-ybbR12 (Yin et al., Proc. Natl. Acad. Sci. USA., 102:15815-15820 (2005)). The PCR fragments were then cloned in to pET14b (NOVAGEN) between the restriction sites of Ndel and BamHI to give the vectors pET14b-EGFP-Sl and pET14b-EGFP-Al which were used to express EGFP as N-terminal fusions to 6xHis tag and C-terminal fusions to Sl and Al tags, respectively.
XV. Labeling S- and A Tagged-EGFP Proteins with Biotin - CoA and ELISA. [00152]Peptide -tagged EGFP proteins were expressed and purified as 6x Histidine fusion proteins by Ni-NTA affinity resin according to standard protocols. PPTase- catalyzed biotin CoA labeling of the purified EGFP proteins was performed according to the procedure previously described (Yin et ah, Proc. Natl. Acad. ScL USA , 102:15815- 15820 (2005)). In a total volume of 100 μL, 0.5 μM Sfp or Acps, 5 μM biotin - CoA, 10
mM MgCl2 in 50 mM HEPES, pH 7.5 were incubated at 37°C with 10 μM S- or A
peptide-tagged EGFP protein for 15 minutes. In order to detect biotin labeling, 20 μL labeling reaction mixture was added to 80 μL 1% BSA in TBS in the wells of a streptavidin coated 96 well plate. Cross-plate series dilution of the labeling reaction mixtures were performed similar to phage ELISA and the plate was incubated at room temperature for 60 minutes and washed five times with 0.05% (v/v) Tween 20, 0.05% (v/v) Triton X-100 in TBS and five times with TBS. Biotinylated EGFP bound to the streptavidin surface were detected by incubation with 1/200 diluted mouse anti-GFP antibody (SANTA CRUZ BIOTECHNOLOGY) followed by incubation with 1/5000 diluted goat anti-mouse IgG antibody HRP conjugate. The plate was then developed by ! adding 100 μL TMB peroxidase substrate and hydrogen peroxide mixture (PIERCE) to each well and incubated at room temperature for five minutes before photography. Biotin labeling of S and Al tagged EGFP in the cell lysates and streptavidin pull down of biotinylated EGFP was performed as previously reported . (Id. Yin et al.)
XVI. Transient Transfection.
[00153]One vial of transfection medium was prepared for each 35-mni well in a 6-well plate. Each vial contained 3 μl of FuGENE 6 transfection reagent (ROCHE DIAGNOSTICS CORPORATION) and 2 μg of the relevant plasmid diluted in 100 μl of Dulbecco's modified Eagle medium/nutrient mixture F-12 (Ham) (DMEM/F12) (1:1). The transfection medium was mixed and allowed to come to equilibrium over an additional 20 minutes. TRVb cells were grown on sterilized coverslips to 50-60% confluency, incubated with transfection medium for 5 hours, and then incubated in standard cell media (with serum) for 24 hours to allow time for protein expression.
XVII. Fluorescent Labeling and Imaging of Al-tagged TfRl.
[00154]TRVb cells transfected with TfRl-Al were incubated in serum-free media for two hours prior to labeling. To label TfRl-Al, cells were then incubated with 1.98 μM Acps and 1 μM CoA-Alexa Fluor 488 in serum-free media for 30 minutes at 37°C. Labeled cells were washed three times with PBS, then incubated with 10 μg/ml Alexa Fluor 568-conjugated Tf (MOLECULAR PROBES INC.) for 5 minutes and washed three times with PBS. Finally, cells were fixed using a 3.7% formaldehyde solution in
PBS, and mounted with SLOWFADE® Antifade Kit (MOLECULAR PROBES INC.)
for optical microscopy studies. Confocal microscopy analysis of samples was performed using a Nikon TE2000U inverted microscope in conjunction with a PerkinElmer Ultraview spinning disk confocal system equipped with a Hamamatsu Orca ER Cooled-CCD camera. Images were acquired using a 6OX differential interference contrast (DIC) oil immersion objective lens, and analyzed using Metamorph software from Universal Imaging, Inc. In order to achieve projections of optical slices, acquired images were processed by MetaMorph using 2D no-neighbors deconvolution followed by 3D reconstruction.
Example 1 : vbbR is a substrate for Sfp
[00155]It has been previously reported that PCP displayed on the surface of M13 phages can be specifically labeled with biotin using Sfp catalyzed transfer {Id., at Yin et al., 2004). In an effort to identify proteins which may be modified by Sfp catalyzed posttranslational modification in the B. subtilis proteome, a genomic library of B. subtilis was displayed on the surface of Ml 3 phages as pill fusion proteins and selected for Sfp catalyzed biotin - Ppant modification. Phages displaying proteins recognized by Sfp for posttranslational modification were covalently labeled with the biotin - Ppant group and selected by binding to immobilized streptavidin. The proteins subjected to Sfp catalyzed posttranslational modification were subsequently identified by sequencing the selected phage clones. The sequence alignment of the various ybbR clones selected by phage display is depicted in Figure 2.
[00156] Among the selected clones, the truncated forms of the predicted ybbR protein corresponding to amino acids 95-278 (JY565), 111-278 (JY503), 214-278 (JY530) and 229-278 (JY529) were selected multiple times by Sfp catalyzed biotin labeling followed by streptavidin binding. In order to confirm that streptavidin binding was indeed dependent on biotin transfer by Sfp-catalyzed reaction, phages expressing various lengths of the displayed ybbR protein were labeled in the presence of both Sfp and biotin — CoA and added to a 96-well streptavidin plate in a phage enzyme linked immunosorbant assay (ELISA). After washing, the phages which bound to a streptavidin plate were detected by anti-M13 phage antibody conjugated with horseradish peroxidase (HRP). All ybbR-displayed phages from the labeling reaction showed greater than a 125 fold streptavidin binding level relative to the control phages where either biotin - CoA or Sfp was excluded during the labeling process (data not shown), suggesting that the various lengths of the ybbR protein displayed on the phage surface as pill fusions can be recognized as substrates of Sfp for biotin labeling. Further, JY529, the shortest ybbR truncated protein selected by phage display, was subcloned into pET21b expression vector and the 49 amino acid residue peptide was
expressed as a fusion protein containing a C-terminal 6x Histidine tag (JY529-His). The
purified protein was incubated with Sfp and biotin - CoA, and the biotin labeling of JY529-His was confirmed by Western blot analysis with streptavidin - HRP conjugate while control reactions with either no Sfp or with no biotin - CoA showed no labeling on the Western blot, as depicted in Figure 3. This suggested that the truncated ybbR proteins were selected by phage display due to their posttranslational modification by Sfp-catalyzed biotin - CoA labeling.
Example 2: Serine274 in ybbR protein is the site of Ppant modification by
Sfp
[00157]The site of Ppant modification in ybbR JY529-His was identified by Fourier- Transform Mass Spectroscopy (FTMS). Truncated ybbR protein JY529-His was covalently modified by Ppant group in presence of CoA and Sfp. The unmodified (apo) and Ppant modified (holo) forms of JY529-His were separated by high pressure liquid chromatography (HPLC) and introduced into the mass spectrometer, resulting in the detection of two species of about 8,513.58 Da and 8,853.37 Da in mass, which corresponded to the mass of the apo and holo forms of JY529-His, as depicted in Figure 4a within 0.36 Da and 0.06 Da (42 ppm and 7 ppm), respectively. The 9+ charge state of holo - JY529-His was mass selected, as shown in Figure 4b, and fragmented using three MS/MS techniques.
[00158]The resulting fragment ions from each technique are shown in Figure 4c. The c and z* ions generated by Electron Capture Dissociation localized the modification to Ser268, Ser274 and Ser280 (according to the numbering in full length ybbR). Further dissociation with IRMPD and OCAD produced a series of y-ions, which demonstrated that Ser280 was not covalently modified, as depicted in Figure 4f. However, another series of y-ions portrayed +80 Da mass shifts, consistent with the ejection of the pantetheinate and retention of phosphate, as shown in Figures 4d and 4e, indicating that the covalently modified serine is Ser274.
[00159]Ser274 in JY529-His was subsequently mutated to Ala by site directed mutagenesis and the Ser274Ala mutant of JY529-His was shown as not being labeled with biotin after incubation with Sip and biotin - CoA as shown by Western blot analysis in Figure 3, confirming the FTMS assignment of Ser274 as the Ppant modification site in JY529-His.
Example 3: Characterization of ybbR Peptides as Substrates of Sfp
[00160] JY529 which includes a ybbR tag and a C-terminal βxHistidine tag, did not show any significant sequence homology with any known ACPs or PCPs that are known substrates of Sfp. The site of Ppant modification in JY529-His, i.e., Ser274, was very close to the C-terminus of the protein, in contrast to the Ppant modified Ser in ACPs or PCPs, which is in the middle of the 80-90 amino acid residue protein with 40-45 residues on either side of the Ser. Thus, in order to determine whether the C-terminal peptide of JY569-His encompassing the Ser274 residue could be recognized by Sfp as a substrate, short peptides corresponding to the flanking sequence of Ser274 in JY529-His were synthesized, as depicted in Figure 5 a, and assayed for Sfp catalyzed Ppant modification. The peptides were incubated with Sfp and biotin - CoA and the reaction mixture was assayed by HPLC. Peptide ybbR13 (DSLEFIASKLA) (SEQ ID NO:2) was found to be modified by biotin — CoA in presence of Sfp, as shown by a product peak with a retention time of 21 minutes on HPLC trace 1, shown in Figure 6a. The product formation was dependent on the presence of both biotin — CoA and Sfp since no product was formed when either biotin - CoA or Sfp were excluded from the labeling reaction (trace 2 and 3 in Figure 6 a).
[00161] Further, Matrix - assisted laser desorption ionization mass spectroscopy (MALDI) confirmed that the product of Sfp catalyzed ybbRl 3 modification by biotin - CoA had the same mass as biotin - Ppant conjugated ybbRl 3 ([M+H]+, calculated 2058.0, observed 2058.2). Similarly, peptides of seventeen and thirteen residues in length - ybbRl 1 (GS QDVLD SLEFIASKLA) (SEQ E) NO:27) and ybbR12 (VLD- SLEFIASKLA) (SEQ ID NO:28) included nine residues LEFIASKLA (SEQ ID NO:29) which extended beyond the Ppant modified Ser274 (underlined), and were Biotin- labeled by Sfp, as shown by HPLC and MALDI (data not shown), hi contrast, peptides with fewer than seven amino acid residues beyond Ser274, e.g., ybbR3 (GSQDVLDSLEFI) (SEQ ID NO:30), ybbR8 (DVLDSLEFI) (SEQ ID NO:31)and ybbR14 (VLDSLEFIAS) (SEQ ID NO:32), were not Biotin-labeled in a Sfp catalyzed reaction, denoting the importance of the C-terminal residues in ybbRll-13 for Sfb recognition.
[00162]Interestingly, the sequence of the last five residues ASKLA (SEQ ID NO:33) at the C-terminus of the Sfp substrates ybbRl 1 - 13 was not encoded by the ybbR ORF in B. subtilis but is part of the sequence between the last residue of truncated ybbR protein
Ile278 and the 6χ Histidine tag in JY529-His. Similarly, in the selected phage clones,
peptide sequence ASKLG (SEQ ID NO:34 ) was part of the linker between residue Ile278 and pill capsid protein. Peptide ybbR15 (VLDSLEFIDGVSL) (SEQ ID NO:35) which had the original ybbR sequence flanking the Ppant modified Ser274 and the same number of residues beyond Ser274 as in Sfp active peptides ybbRl 1-13, failed to be the substrate of Sfp, suggesting the full length ybbR protein in B. subtilis proteome is not a substrate of Sfp.
[00163]Before carrying out a detailed kinetic analysis of ybbR peptide modification by Sfp, the activities of Sfp catalyzed ybbRl 3 modification by biotin - CoA were determined at various pHs ranging from 5.0 to 8.5 and it was found that Sfp has the highest activity between pH 7.0 and 8.0, thus all kinetic measurements were done at pH 7.5. Figure 6b shows a typical Michaelis - Menten plot for the Sfp catalyzed ybbR peptide modification reaction. Detailed kinetic analysis was carried out for the Sfp catalyzed modification of peptides ybbRl 1-13 by biotin - CoA and fluorescein - CoA, as summarized in Table 1. Peptides ybbRll - 13 have the same nine amino acid residues (LEFIASKLA) (SEQ ID NO:29) C-terminus to Ppant modified Ser274 but have a different number of amino acid residues N-terminus to the Ppant modified Ser274, for example, seven amino acid residues in ybbRll, three amino acid residues in ybbR12 and only one amino acid residue in ybbR13. Notably, all three peptides showed
similar Kn, (122.8 - 141.7 μM) andkcat (9.3 - 12.1 min"1) values for Sfp catalyzed
peptide modification at saturating biotin— CoA concentration (150 μM), as shown in
Table 1, suggesting that the N-terminal sequence of the ybbR peptides does not play a significant role in Sfp recognition.
[00164] Comparison to the GrsA-PCP with a Km of 4.1 μM and a kcat of 10.3 min"1 for
Sfp catalyzed biotin - CoA loading shows that peptides ybbRl 1-13 have a 30 fold higher Km and similar kcat values, which maybe due to the larger surface area of PCP that can interact with Sfp. Similar values of Km and kcat for biotin - CoA were found' at saturating
concentrations of ybbRl 3 peptide (500 μM) and PCP (20 μM), respectively, suggesting
that the binding of either PCP or peptide as substrates to Sfp did not affect the binding and the turnover of biotin - CoA.
[00165]HPLC and MALDI analysis also confirmed that peptides ybbRl 1-13 can be modified by fluorescein - CoA in the presence of Sfp, suggesting that Sfp retained its substrate promiscuity with various small molecule probes conjugated to CoA for the site specific modification of short ybbR peptides. The ybbRl 3 peptide was modified by fluorescein - CoA at a similar rate as biotin - CoA, with a Km of 69.9 μM and a kcat of 19.1 min"1, as summarized in Table 1, suggesting that Sfp does not differentiate between the various small molecule probes conjugated to CoA. Table 1 Kinetic parameters of Sfp catalyzed ybbR tags and PCP modification
Figure imgf000073_0001
Example 4: Characterization of the ybbR peptides using Circular Dichroism
[00166]Peptides ybbRl 1 - 13 were aligned with PCPs or ACPs from known NRPS and PKS modules: PksL (Kunst et al. Nature 390:249-256, 1997); TycC3 (Weber at al. Structure FoI Des. 8: 407-418, 2000); EntB (Gehring et al., Biochem. 36:8495-8503, 1997); GrsA (Stachelhaus et al. Biochem. 39: 5775-87, 2000); HMWP2 (Keating et al., Biochem. 39: 4729-4739, 2000); and ACP from FrenN (Li et al, Biochem. 42:4648-57., 2003) and a conserved DSl, tripeptide sequence motif was identified at the Ppant modified Ser (underlined), as depicted in Figure 5a.
[00167]NMR structural studies on TycC3-PCP {Id., at Weber et al) and FrenN-ACP (Id. at Li et al.) suggested that PCP and ACP adopted a similar anti-parallel four-helix bundle fold with the Ppant modified Ser at the end of a long flexible loop and immediately followed by helix II, as depicted in Figures 5b and 7. Since the nine residue sequence LEFIASKLA (SEQ ID NO:29) following the Ppant modified Ser274 in ybbR peptides 11-13 was found to be the key element for Sip recognition and could be mapped to helix It in the known structures of PCP and ACP, it was determined
whether the ybbR peptides could form an α-helix in the solution in order to be
recognized by Sfp as substrates. The ybbR peptides previously tested for Sfp catalyzed modification were dissolved in 5 niM potassium phosphate buffer, pH 7.5 with 30% 2,2,2-trifluoroethanol (TFE) and the peptide conformation was measured by circular dichroism (CD) spectroscopy. The CD spectra of Sfp active peptides ybbRl 1, ybbR12 and ybbRl 3 all showed two minima at 208 nm and 222 nm and an isodichroic point
close to 200 nm, which are characteristics of an α helical conformation, shown in Figure
8a. (Greenfield et al., Biochem. 8:4108-4116, 1969). The α-helix content of these peptides in aqueous TFE were estimated to be 57.3% for ybbRl 1, 36.0% for ybbR12 and 35.1% for ybbR13. (Id.) In contrast the Sfp inactive peptides ybbR3, ybbR8,
ybbRl 4 and ybbRl 5 exhibited a lesser extent of α-helical structure and more undefined
structure with the α-helix content of these peptides was estimated to be 16.6%, 11.9%,
16.6% and 7.7%, respectively, as shown in Figure 8b, suggesting that the C-terminal residues ASKLA (SEQ ID NO:33) in peptides ybbRl 1, 12 and 13 were important for
the propensity of these peptides to adopt α-helical conformation in solution.
[00168]Since peptides ybbRl 1 - 13 all had a strong tendency for α-helix formation in
solution and were good substrates of Sfp, small peptides with sequences flanking the Ppant modified Ser and encompassing helix II in known PCPs and ACPs were tested as potential substrates of Sfp. Based on the sequences of TycC3-PCP (Id. at Weber et al.) and PksL-ACP (Id. at Kunst et al), two peptides TycC3 (GGHSLKAMAVAAQVHREY) (SEQ ID NO:36) and PksLl (GLNSSGLLEVVETISDKI) (SEQ ID NO:37) were synthesized with the Ppant modified Ser (underlined) at the fourth residue followed by eleven residues involved in helix II, similar to the arrangements in ybbR12 shown in Figure 5 a. Although the CD
spectra of peptides TycC3 and PksLl both showed significant α-helical content (22.49%
and 36.91% by estimation) (shown in Figure 8c), no biotin - Ppant modified peptides were identified by HPLC or MALDI after overnight incubation of the peptides with Sfp and biotin - CoA at various pHs ranging from 5.0 to 8.5. Therefore peptides with sequences encompassing helix II in the PCP or ACP domains were determined not to be substrates of Sfp.
Example 5: ybbR peptides can be used for site-specific labeling of proteins [00169]In order to determine whether Sfp active ybbR peptides could be used as tags for site specific protein labeling, 13 residue ybbR12 peptide (VLDSLEFIASKLA) (SEQ ID NO:37) was fused either to the N- terminus or the C-terminus of enhanced green fluorescent protein (EGFP), glutathione-S-transferase (GST) and maltose binding protein (MBP), and the purified fusion proteins were labeled with biotin-Ppant in the presence of Sfp and biotin — CoA. The covalent labeling of the ybbR tagged target proteins by biotin was confirmed using ELISA by binding various biotin labeled target proteins to streptavidin plates and probing with HRP conjugated antibodies against the target proteins (data not shown). Western blots probed with streptavidin - HRP also showed that ybbR tagged EGFP, GST and MBP were labeled with biotin after a 15 minute incubation with biotin - CoA and Sfp (data not shown). [0017O]In order to determine whether the labeling reaction can be carried out in cell lysates, a 1 ml E. coli overnight culture was lysed using sonication followed by the addition of biotin-CoA and Sfp to the lysates. After 15 minutes, the labeling reaction mixture was prepared for polyacrylamide gel electrophoresis (PAGE) under reducing conditions after which the protein was transferred to PVDF membrane and blots were probed with streptavidin - HRP conjugate. Biotin labeling of all ybbR tagged fusions in the cell lysates was confirmed by Western blot analysis and the control reactions where either biotin - CoA or Sfp was excluded did not give any biotin labeling, as shown in Figure 9, nor did the control reactions in which target protein without the ybbR tag was incubated with biotin - CoA and Sfp (data not shown), suggesting that the labeling reaction is strictly dependent on Sfp catalyzed biotin Ppant transfer onto Ser274 in the ybbR peptide.
[00171] Shorter ybbR peptide were fused to target proteins and tested for labeling by Sfp catalyzed reaction. An eleven amino acid residue peptide tag ybbR13 (DS.LEFIASKLA) (SEQ ID NO:2) was fused either to the N-terminus or the C-terminus of EGFP, and the fusion protein was labeled using Sfp catalyzed biotin - CoA transfer, as shown in Figure 9. When a six residue peptide DS-LEFI (SEQ ID NO:19) with a truncated C-terminus was fused to the C-terminus of EGFP, GST or MBP, none of the fusion proteins were labeled with biotin Ppant as shown by ELISA and Western blot. However, target proteins with the same peptide sequence fused to the N-terminus of the proteins were labeled with biotin by Sfp catalyzed biotin - CoA transfer, as shown in Figure 9. This suggests that residues ASKLA at the C-terminus of ybbR13 (DSLEFIASKLA) (SEQ ID
NO:2) were crucial for the formation of α-helical conformation and subsequent Sfp
recognition when the ybbR peptide was at the C-terminal end of the protein. In contrast when the ybbR peptide was at the N-terminus, the presence of residues ASKLA (SEQ ID NO:33) in the ybbR peptide might not be necessary since the peptide was followed
by the N-terminal residues of the target proteins, which are thought to facilitate α-helical
formation of the N-terminal ybbR peptide for recognition by Sfp. [00172]In order to determine whether the ybbR peptide is a substrate of Sfp when inserted in the middle of the target protein, a ybbR peptide with flanking glycine residues (GGGTVLDSLEFIASKLAGGG, (SEQ ID NO:38) was inserted in place of Tyrl45 of EGFP to give the construct EGFP-ybbR145, as shown in Figure 10a, since it has been previously reported that calmodulin or zinc finger proteins inserted at the same position in EGFP did not effect protein fluorescence. (Baird et al, Proc. Natl. Acad. Sci. USA, 96:11241-46, 1999). Biotin labeling of EGFP-ybbR145 by Sfp, either as a purified protein or in cell lysates, was confirmed by Western blot analysis, as shown in Figure 9, and the EGFP-ybbR protein was labeled site specifically with tetramethylrhodamine and Texas Red upon Sfp catalyzed fiuorophore - CoA transfer onto the internal ybbR peptide in EGFP-ybbR145, as shown in Figure 10b.
Example 6: Location of the vbbR peptide does not effect the yield of the labeled protein
[00173]To quantify the yield of the protein labeling reaction, various EGFP proteins shown with the ybbR tag fused to the N or C terminus or inserted in the middle of the protein were labeled with biotin after incubation with Sfp and biotin-CoA for 30 minutes. Specifically, 23.5 μM of an EGFP protein with a ybbR tag at the N or the C terminus or in the middle of the protein was incubated for 30 minutes at 370C with 50 μM biotin-CoA and 0.5 μM Sfp in 10 mM MgC12, 50 mM HEPES, pH 7.5 buffer followed by the addition of 0.3 mL UltraLink Immobilized Streptavidin Gel (PIERCE) with a biotin binding capacity of 100 nmol/mL gel. After a 30 minute incubation at room temperature on a rotating platform, the tubes were centrifuged and the UV absorbance at 490 nm of the supernatants were measured in order to quantify the concentration of the EGFP proteins in the solution. For control, either no Sfp was added to the labeling reaction mixture or N-terminal PCP-EGFP fusion protein was used for biotin labeling and subsequent streptavidin beads immobilization. As depicted in Figure 11, all of the EGFP proteins had similar yields, regardless of the location of the ybbR peptide. Example 7: Identification of A series and S series of peptides as novel substrates of phosphopantetheinyl transferases
[00174] As discussed above, PCP and ACP domains have a conserved (H/D)S(L/I) motif at the site of Ppant modified Ser (underlined) with the residue preceding the conserved Ser as a His or Asp and the residue following the Ser a Leu or He (Marahiel et al., Chem. Rev., 97:2651-2674 (1997)). Therefore, for the peptide library to be displayed on the phage surface, the DS sequence in the original ybbR peptide was retained. A Leu or He amino acid residue was maintained at the site immediately following the Ppant modified Ser by using a combination of CTT and ATT codons in the library. The remainder of the eight residues were completely randomized by the use of NNK codons (N = A or C or G or T; K = G or T). Primers were synthesized for the cloning of the peptide library encoding the sequence GDS(L/I)XXXXXXXX (X = any of the 20 amino acid residues) (SEQ ID NO.53), inserted into the pComb3H plasmid (Barbas et al., Proc. Natl. Acad. Sci. USA., 88:7978-82 (1991)), and the peptide library was subsequently displayed on the surface of M 13 phage as N-terminal fusions to the phage capsid protein pill. The final size of the library was approximately 1 x 109. [00175]The peptide library was selected in parallel in separate tubes by Sfp- and Acps- catalyzed biotin-Ppant attachment to the phage-displayed peptides using biotin-SS-CoA as the substrate, as shown in Figures 12b and 12c. After the first round of selection, the library diverged in the subsequent rounds, in that phages selected by Sfp were exclusively used for the next round of Sfp selection and phages selected by Acps were exclusively used for the next round of Acps selection. For the first round of selection, 5 μM biotin-SS-CoA, 1 μM Sfp or 5 μM Acps were used to biotinylate 1012 phage particles in a total volume of 1 mL for 1 hour at 30°C. As the selection process proceeded, the concentration of biotin-SS-CoA, enzymes Sfp or Acps and the density of phage particles were decreased round by round to increase the stringency of the selection. Eventually, in the fifth round of selection, only 1 μM biotin-SS-CoA, 0.08 μM enzymes Sfjp or Acps and 1010 phage particles were incubated for 7 minutes at 300C. During the selection, after the enzyme-catalyzed biotin labeling reaction, phage particles were precipitated by polyethylene glycol (PEG) to quench the reaction and remove unreacted biotin-SS-CoA followed by resuspension in 1% bovine serum albumin (BSA) in Tris buffered saline (TBS), pH 7.4. The phage particles displaying peptides as the substrates of Sfp or Acps were covalently conjugated to biotin through the Ppant arm linked to a disulfide bridge and subsequently the phages were allowed to bind to streptavidin-coated 96 well plates, as shown in Figure 12c. After washing, biotin- conjugated phage particles were cleaved from the solid support by incubating with 20 mM dithiothreitol (DTT) in TBS. The eluted phage particles were then rescued by infecting E. coli XLl -Blue cells and carried on for the next round of selection. [00176] In parallel to the selection reaction with Sfp and Acps, controls were performed excluding the enzymes or biotin-SS-CoA in the labeling reaction before binding the phage particles to the streptavidin plates. There was a steady increase in the ratio of phage recovery for the reaction with the addition of both the enzyme and biotin-SS-CoA over the controls, as depicted in Figure 13. For the fifth and last round of selection, the ratio of the reaction over the controls was more than 104, suggesting the selection of phage particles by streptavidin binding was indeed dependent on the enzyme Sfp or Acps to catalyze biotin labeling of the phage displayed peptides and that the peptide clones enriched after the fifth round of selection could be very efficient substrates of Sfp and Acps.
[00177] After the fifth round of selection, forty clones from either the Sfp-selected library or Acps-selected library were sent for DNA sequencing and significant sequence convergence for the peptide being selected was observed, as shown in Figure 14. Table 2 lists the designations and the peptide sequences of the most abundant phage clones after the fifth round of selection and Figure 15 shows the degenerate peptide sequences that were selected by Sfp or Acps with the preferred residues at each position. As shown in Table 2 and Figure 16, peptide clone Sl and a closely related sequence Sl' with two mutations appeared a total of 6 times among the 30 sequencing samples from the Sfp-selected libraries; similarly, peptide clone Al was counted 8 times among the 40 sequencing samples from the Acps-selected library. This suggested that Sl and Al clones started to dominate the final selected pool of peptides after the fifth round of selection using Sfp and Acps, respectively, and thus the phage selection was stopped at the fifth round.
Table 2. Summary of the selection results. "Number of occurrences" designates the number of times the same peptide clone was identified within the phage clones sent for DNA sequencing after the fifth round of selection. Ppant modified Ser is underlined.
Sfp selected phage clones
Name Peptide sequence Number of occurrences
Sl GDSLSWLVRCLN (SEQ ID NO:54)
Sl' GDSLSWLLRCLH (SEQ ID NO:55)
S4 GDSLSYMLSLIY (SEQ BD NO:56)
S5 GDSLTWMTWMME (SEQ ID NO:57)
S9 GDSLSWLSLLLQ (SEQ ID NO:58)
Asps selected phage clones Name Peptide sequence Number of occurrences
Al GDSLDMLEWSLM (SEQ YD NO:41)
A2 GDSLDWMSCIDY (SEQ ID NO:59)
A3 GDSLDLLSYCLG (SEQ ID NO.-60)
A4 GDSLWQLEMAM (SEQ ID NQ:61) Example 8: Characterization of the enriched phage clones.
[00178]In order to verify that the enriched phage clones did indeed display peptides that were the substrates of Sfp and Acps, the most abundant phage clones from the fifth round selection identified by DNA sequencing — Sl, S4, S5, and S9 from the Sfp selection and Al, A2, A3, and A4 from the Acps selection — were propagated separately and subjected to a phage enzyme-linked immunosorbent assay (ELISA), as depicted in Figure 17. In separate tubes, phages displaying different peptide sequences were labeled with biotin by either Sfp or Acps using biotin-CoA as the donor of the biotin-Ppant group. Control reactions were also run in parallel with the exclusion of the enzymes or both the enzymes and biotin-CoA. After the labeling reaction, the reaction mixtures were added to the streptavidin-coated 96 well plate and diluted across the plate by 5-fold from column to column to allow the binding of biotin conjugated phage i particles to the streptavidin surface. After washing, phages retained in each well were detected using an anti-M13 phage antibody conjugated to horseradish peroxidase (HRP). [00179]Figure 16 depicts the phage ELISA results for the two most abundant phage clones — Sl enriched by Sfp selection and Al from Acps selection. While ELISA gave very good signals for biotinylated phage binding to the streptavidin plate in the fourth column after 125-fold dilution of the labeling reaction mixture (Figure 16, rows 1 and 6), the corresponding phage binding of the control reactions with no biotin-CoA or enzymes added was very low in the first column. Therefore, both Sl and Al peptides displayed by phage particles could be modified very efficiently by Sfp or Acps, and the enrichment of those clones through the five rounds of selection was strictly dependent on Sfp- or Acps-catalyzed biotin labeling of the displayed peptides. [00180]Phage ELISA also showed the results when Sl peptide-displayed phages were incubated with Acps and biotin-CoA or, vice versa, Al peptide-displayed phages were incubated with Sfp and biotin-CoA (Figure 16, rows 2 and 5). In these cross-tests, the levels of phage biotin labeling were very low, as suggested by the low ELISA signals that were almost the same as the control reactions with no biotin-CoA or enzymes added. This suggested the Sl peptide selected by Sfp selection was a poor substrate for Acps and likewise the Al peptide selected by Acps was a poor substrate for Sfp. Other phage-displayed peptide clones S4, S5 and S9 from the Sfp selection and A2, A3 and A4 from the Acps selection showed similar results — high level of biotin labeling with the PPTase used for selection and background level of biotin modification with the other PPTase for the cross-labeling (Figure 17). Accordingly, using the selection strategy as described herein, two series of peptides: the S peptides (Sl, S4, S5 and S9) with preferred substrate specificity with Sfp and the A peptides (Al, A2, A3, A4) with preferred substrate specificity with Acps, were identified. Furthermore, these two series of peptides did not share a great deal of sequence homology except for the DSL sequence flanking the Ppant modified Ser (underlined), suggesting the S and A series of peptides could potentially serve as orthogonal tags for protein labeling by Sfp and Acps PPTases.
Example 9: Kinetic characterization and mutagenesis studies of the selected peptides.
[00181 ]In order to measure the kinetics of the serine residue posttranslational modification reaction, peptides Sl, S4, S5, S9 identified from the Sfp selection of the phage-displayed peptide libraries and peptides Al , A2, A3 and A4 from the Acps selection were synthesized as 12-mers, all starting with the GDSL sequence flanking the Ppant modified Ser (underlined). Sequence alignment data revealed that one of the S series of peptides, e.g., Sl', selected by Sfp selection, differed from the Sl peptide at position 8 as it had a Leu instead of a VaI amino acid residue (Ppant modified Ser is designated position 3). Peptide S2 was synthesized as having a VaI to Leu mutation at position 8. It was speculated that because peptide Sl has a Cys residue at position 10, that the Cys residue may introduce complexities for disulfide formation, should Sl be used as a tag for protein labeling. Accordingly, peptide S3 was synthesized with a Cys to Leu mutation at position 10 in the Sl peptide and S6 was synthesized with the same mutation in the S2 peptide, as depicted in Table 3. The amino acid residue Cys was replaced by Leu because some peptides selected by Sfp had a Leu residue at the same position, as shown in Figurel4.
[00182]The kinetic measurements of the biotin Ppant transfer reaction from biotin-CoA to the various peptides were performed with pure Sfp or Acps, and the results are depicted in Table 3. The Sl, S2 and S3 peptides showed similar specific activity for Sfp-catalyzed peptide modification by biotin-CoA, with a kcat/Km in the range of 0.029 to 0.083 μlVrWn"1, comparable with the kcat/Km of 0.091 μM'Wn"1 for the ybbR tag. The specific activity of the S6 peptide (kcat/Km=0.19 μM'Wn"1) was twofold higher than the ybbRl 3 peptide, mainly contributed by a lower Km of 51.5 μM, compared to a Km of 123 μM for the ybbRl 3 peptide. However, when the Cys at position 10 in peptide S2 was mutated to Ser to give peptide S7 instead of Leu as in S6, S7 had a significantly higher Km of 221 μM, suggesting the Leu residue at position 10 is important for Sfp binding of the substrate peptide.
[00183]The Al peptide showed activity for Acps-catalyzed peptide labeling, with a kcat/Km of 0.015 μM^min"1 and a Km of 117 μM, comparable to the activities of the S peptides for Sfp-catalyzed protein labeling. Corresponding peptides from the phage clones enriched by Sfp selection, S4, S5 and S9, and from Acps selection, A2, A3, and A4 were also synthesized. Their specific activities with Sfp and Acps respectively were more than 5- to 10-fold lower than the S6 and Al peptides, consequently, detailed kinetic characterizations were not carried out with them. [00184] Cross-reactivity of the Al peptides with Sfp and S peptides with Acps was also assayed and the results are shown in Table 3. The S peptides were poor substrates for Acps modification with a kcat/Km of around 0.0005 μM'Wn"1. Among the S peptides, the S6 peptide showed the highest level of differentiation for different PPTases, with a 442-fold higher kcat/Km for Sfp-catalyzed peptide labeling than the Acps-catalyzed reaction, hi contrast, the ybbR13 peptide, identified as a Sfp substrate, showed substantial activity with Acps, with a kcat/Km of 0.0033 uM'Wn"1, only 28-fold lower than the Sfp-catalyzed reaction. Therefore, S6 appears to be a better tag for Sfp labeling than ybbR not only because it has a lower Km and higher activity as the substrate of Sfp but also because it shows more than 7-fold lower activity (kcat/Km) than ybbR for Acps- catalyzed modification. Both the S6 and ybbR peptides show a preference for Sfp over Acps; however, the S6 peptide showed a greater than about a 440-fold preference as a substrate of Sfp versus Acps, while the ybbR tag showed a preference for Sfp φf only 28-fold over Acps.
[00185]A1 was a poor substrate for Sfp, with a WKn, of 0.00049 μM'Wn"1, 30-fold lower than that of the reaction catalyzed by Acps. Thus, by selection of phage-displayed peptide libraries and later mutagenesis studies, two additional peptides were identified, S6 and Al, which are good substrates for the peptide labeling reactions catalyzed by Sfp and Acps, respectively. At the same time S6 and Al also show significant orthogonality in enzyme differentiation, with S6 a preferred substrate of Sfp and Al a preferred substrate of Acps. Accordingly, S6 or ybbR and Al could be used for simultaneous labeling of molecules, by simultaneous Sfp and Acps catalyzed reactions, respectively. Table 3. Kinetic characterization of Sφ- and AcpS-catalyzed peptide labeling reaction by biotin-CoA (2). Ppant modified Ser is underlined. Mutations made to the Sl peptide sequence were shown in bold.
Sfp AcpS kcat kcat Km kcat/Km kcat' -^-m
Peptide Sequence (mm" (μM (μM- (mi (μM) (μM- (SfP)/
') ) W-1) n 1) 1EQm"1) kcat/K-m
(AcpS) ybbR13 DSLEFIASKLA 11 123 0.091 0.81 242 0.0033 28
(SEQ ID NO:2)
Sl GDSLSWLVRCL 4.1 139 0.029 0.04 77.2 0.00054 53
N 2
(SEQ ID NO:54)
S2 GDSLSWLLRCLN 10 120 0.083 0.05 108 0.00055 150
(SEQ ID NO:62) 9
S3 GDSLSWLVRLL 3.1 61.8 0.050 0.03 105 0.00032 156
N 4
(SEQ ID NO:63)
S6 GDSLSWLLRLLN 10 51.5 0.19 0.03 76.0 0.00043 442
(SEQ ID NO:40) 3
S7 GDSLSWLLRSLN 8.4 221 0.038 0.08 254 0.00033 115
(SEQ ID NO:64) 5 kcat'j^-m
Figure imgf000085_0001
(SEQ ID NO:41)
Example 10: Use of S6 and Al Peptides for Protein Labeling.
[00186]The S6 and Al 12-mer peptide were evaluated as peptide tags fused to target proteins. The Sl and Al peptides were fused to either the N or C termini of the enhanced green fluorescence protein (EGFP) and tested for biotin labeling with EGFP as the target protein. Later, the optimized S2 and S 6 tags were also fused to the N-terminal of EGFP. As in case of the phage ELISA experiments, Sl-, S2-, S6- and Al-tagged EGFP were tested for biotin labeling by both Sφ and Acps using biotin-CoA as the substrate, and the labeling results were compared to biotin labeling of ybbR- or PCP- tagged EGFP. Control reactions were also run with the exclusion of enzymes or both enzymes and biotin-CoA. The labeling reaction mixture was loaded on the 96-well streptavidin plate and a 5 -fold series dilution was carried out across the wells in the plate. After washing, biotinylated GFP immobilized onto the streptavidin surface was detected using a mouse anti-GFP antibody and a goat anti-mouse antibody-HRP conjugate.
[00187]N-terminal Sl-, S2- or Sό-tagged EGFP proteins were efficiently labeled with biotin by Sfp using biotin-CoA as the substrate. Similarly, N-terminal Al -tagged EGFP was efficiently labeled with biotin by Acps. In contrast, the ELISA signals for Acps- catalyzed biotin labeling of EGFP fused to Sl, S2 or S6 tags were more than 100-fold lower than the same labeling reactions catalyzed by Sfp, suggesting that the S-tagged proteins were not labeled by Acps efficiently. Also, Al -tagged EGFP was not labeled with biotin by Sfp very efficiently, as shown by ELISA. Accordingly, these results demonstrate that the S tags and the Al tag can be used as orthogonal tags for target- specific protein labeling catalyzed by Sfp and Acps, respectively. The S and Al peptide tags were also fused to the C-terminal of EGFP or to the N-terminal of glutathione S- transferase (GST) and maltose binding protein (MBP). All showed similar labeling efficiency, with S-tagged proteins preferentially labeled by Sfp and Al -tagged proteins preferentially labeled by Acps (data not shown), denoting the portability of the S and Al tags for the construction of fusions at N- or C-termini of various target proteins. Figure 18 also shows the biotin labeling of N-terminal ybbR- or PCP-tagged EGFP catalyzed by Sfp or Acps. As expected, PCP-tagged EGFP showed preferential labeling by Sfp, although ybbR-tagged EGFP showed significant labeling by both Sfp and Acps, suggesting that the S tags are more specific as substrates for Sfp-catalyzed protein modification than the ybbR tag. S and Al tagged EGFP can also be labeled with biotin in the cell lysates by Sfp and Acps, respectively, and the biotinylated protein can be detected by ELISA (data not shown). [00188]In order to quantify the yield of the protein labeling reaction, S - or Al -tagged EGFP proteins were labeled with biotin by Sfp and Acps followed by the addition of streptavidin coated agarose beads to pull down the biotin-labeled EGFP. More than 80% of the S- or Al -tagged EGFP could be immobilized on the streptavidin beads after the biotin labeling reaction catalyzed by Sφ or Acps, respectively. On the other hand, less than 5% of the EGFP could be pulled down by streptavidin beads in the cross- labeling reaction using Sφ to label Al -tagged protein or Acps to label S-tagged protein.
Example 11: Cell surface Labeling Using the Al Peptide Tag. [00189]The transferrin-transferrin receptor 1 (Tf-TfRl) system was used to demonstrate surface protein labeling of cells using the Al peptide tag. TfR-mediated iron uptake represents the major mechanism used by vertebrate cells to acquire iron from the environment, and the endocytic cycle of Tf-TfRl has been well established (Klausner et al, Proc. Natl. Acad. Sci. USA, 80:2263-2266 (1983); Morgan, MoI. Aspects Med., 4: 1-123 (1981)).
[00190]The Al peptide tag was attached to the extracellular C-terminus of TfRl, where modifications have been shown to have little effect on the iron-uptake function of the receptor. TfRl-Al was transiently transfected into TRVb cells, a Chinese hamster ovary cell line that lacks endogenous TfRl, and labeled with CoA-conjugated Alexa Fluor 488 in the presence of Acps. Transfected cells were then incubated with Alexa Fluor 568 diferric human transferrin conjugates (Tf- Alexa 568) for 5 minutes, and fixed for observation under the confocal microscope. Imaging of transferrin receptor 1 using the Al peptide tag was performed. TRVb cells were transfected with TfRl-Al and then incubated with AcpS and Co A- Alexa Fluor 488 for 30 minutes, washed, and incubated with Alexa Fluor 568-labeled transferrin for 5 minutes. Cells were fixed and imaged using confocal microscopy. Stacks of optical slices at 0.25 μm per step were processed by MetaMorph using 2D no-neighbors deconvolution followed by 3D reconstruction. Colocalization of TfRl-Al and Tf was observed (data not shown). [00191 ] Accordingly, these results demonstrate that the Al peptide tag may be used for surface protein labeling in mammalian cells in order to monitor the interactions and trafficking of cell surface receptors while allowing these receptors to maintain their cellular function.
[00192]C-terminal Sl-, S2- and Sό-tagged TfRl proteins were also constructed and tested for labeling by Alexa Fluor 488 in the presence of Sfp. Although labeling of TfRl on the cell surface was observed, the yield of the labeling reaction was significantly lower than that, of the PCP-tagged transferrin receptor (data not shown), suggesting the S tags fused to TfRl may not be as well exposed on the cell surface as the Al tag or PCP tag for efficient protein labeling.
Example 12: Comparison of the S, A and vbbR peptides from the phage selection for Sfp and Acps Modification.
[00193] As discussed above, two short peptide tags S6 and Al were identified as orthogonal substrate sequences for Sfp- and Acps-catalyzed peptide tag labeling reaction. The circular dichroism (CD) spectrum of peptide S6 indicates a tendency to adopt an α-helical conformation in 30% TFE similar to the previously reported Sfp substrate ybbR13 while the features for an α-helical conformation in the CD spectrum of the Sl peptide are not as significant (Figure 19). These results suggest S peptides may adopt an α-helical conformation and mimic helix II in PCP upon their binding to Sfp. The sequences of Sl, S6 and ybbR in a helical wheel representation (Figure 20) were compared and it was found that Sl, S6 and ybbR13 bear significant similarity despite their difference in primary amino acid sequences. S6, the peptide that showed highest reactivity with Sfp, retains the Leu residues at positions 4, 7, 8 and 11 as does the ybbR peptide with Leu, He, Ala and Leu at the corresponding positions (Ppant modified Ser is designated position 3). Those residues form the hydrophobic side of the α-helix shared by both ybbR13 and S6. Also the ybbR13 peptide has a Phe residue at position 6 and correspondingly phage selection of the peptide library in this work leads to an aromatic Trp residue at the same position in S6. The ybhR13 peptide has a basic Lys residue at position 10 while the current, independent selection leads to an Arg side chain at position 9 in close proximity in S6 peptide. Overall, S6 and ybbR13 share very similar organization for the assembly of α-helix and since the hydrophobic side of the helix is preserved in the selection. It is contemplated that the helix mimics the neutral protein surface of PCP upon the interaction of peptides S6 and ybbR13 with Sfb. S6 was derived from peptide Sl, which was the most abundant clone enriched by phage selection using Sfp. Two mutations in Sl, VaI to Leu at position 8 and Cys to Leu at position 10, gives S6 which is kinetically a more active substrate for Sfp. These mutations are both on the hydrophobic side of the helix and give a condensed cluster of Leu side chains on this side of the α-helix. Correspondingly, S6 has a twofold lowering of Km compared to ybbR13 and Sl peptides and a higher tendency to adopt an α-helical conformation than Sl, suggesting the formation of the hydrophobic face of the α-helix may well contribute to the binding of S6 peptide to Sfp and the subsequent peptide modification reaction.
[00194]The Al peptide does not have significant α-helical conformation in 30% TFE according to its CD spectrum (Figure 19). Without wishing to be bound by theory, it is, nevertheless contemplated that the Al peptide forms an α-helical conformation upon binding to Acps.
[00195]A helical wheel plot of the Al peptide shows a very different helical surface from those of the Sl, S6 and ybbR13 peptides (Figure 20). Although Al also has Leu side chains at positions 4, 7, 11- which may promote helix formation, Al has negatively charged Asp and GIu side chains at positions 5 and 8, respectively, and the polar residue Ser at positions 3 and 10, which makes the hydrophobic side of Al considerably smaller. It is contemplated that this property may make the Al peptide a poor substrate of Sfp. On the other hand, since Acps is a highly positively charged protein with a pi value of 9.6 (Id. at Parris et al.), those residues may be contributing to the binding of peptide Al to Acps by mimicking the highly negatively charged surface of ACP. [00196] Accordingly, because S6 and Al peptides are quite different in electrostatic distribution on putative formation of an α-helix, and this may be the structural basis for the orthogonality in reactivity of those peptides as modified by Sfp versus Acps. [00197] The specification is most thoroughly understood in light of the teachings of the references cited within the specification which are hereby incorporated by reference. The embodiments within the specification provide an illustration of embodiments in this disclosure and should not be construed to limit its scope. The skilled artisan readily recognizes that many other embodiments are encompassed by this disclosure. All publications and patents cited and sequences identified by accession or database reference numbers in this specification are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with the present specification, the present specification will supercede any such material. The citation of any references herein is not an admission that such references are prior art to the present disclosure.
[00198]Unless otherwise indicated, all numbers expressing quantities of ingredients, cell culture, treatment conditions, and so forth used in the specification, including claims, are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters are approximations and may very depending upon the desired properties sought to be obtained by the present invention. Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:
1. A method of generating a molecule of formula (I) :
Mq-L-Y-Z (I) wherein:
M is a first agent; q is an integer greater than 0;
L is a linking group;
Y is an alpha-helical oligopeptide moiety; and Z is a second agent; comprising: a) providing a compound of formula (II):
Y-Z (II) wherein:
Y is the alpha-helical oligopeptide moiety; and Z is the second agent; and b) contacting said compound of formula (II) with an enzyme having phosphopantetheinyl transferase activity or a fragment thereof having phosphopantetheinyl transferase activity, in the presence of a compound of formula (III):
Mq-L-N (III) wherein:
M is the first agent; q is an integer greater than 0; L is the linking group; and N is a leaving group; under suitable conditions to allow the enzyme or the fragment thereof to attach the linking group (L) to the alpha-helical oligopeptide moiety (Y), thereby generating the compound of formula (I).
2. A molecule of formula (IV) :
Mq-L-Y-Z (IV) wherein:
M is a first agent; q is an integer greater than 0;
L is a linking group;
Y is an alpha-helical oligopeptide moiety; and
Z is a second agent.
3. The molecule of claim 2, wherein Mq-L- is of the formula (V):
Figure imgf000093_0001
wherein each M is an independently selected first agent for each occurrence.
4. The molecule of claim 2, wherein Mq-L is of the formula (VI):
Figure imgf000093_0002
wherein (VI) each M is an independently selected first agent for each occurrence.
5. The molecule of claim 2, wherein said linking group is capable of being attached to the alpha-helical oligopeptide moiety via a phosphopantetheinylation reaction.
6. The molecule of claim 2, wherein the first agent is chosen from the group consisting of a small molecule moiety, a proton, a linker group, an affinity probe, a spectroscopic probe, a radioactive probe, a peptide, a nucleic acid, a lipid molecule, a bio-molecule, a radical generating molecule, a toxin, a polymer, a receptor binding molecule, a sugar, or an antibody.
7. The molecule of claim 6, wherein the small molecule moiety is a drug moiety.
8. The molecule of claim 6, wherein said spectroscopic probe is chosen from a fluorophore, a chromophore, a magnetic probe and a contrast reagent.
9. The molecule of claim 6, wherein said affinity probe is chosen from biotin, glutathione.
10. The molecule of claim 2, wherein the second agent is a polypeptide comprising an amino acid sequence which is different from the amino acid sequence of the oligopeptide moiety.
11. The molecule of claim 10, wherein said alpha-helical oligopeptide moiety forms an alpha-helical structure when located at the N-terminus of the polypeptide.
12. The molecule of claim 10, wherein said alpha-helical oligopeptide moiety has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:40 and SEQ ID NO:41.
13. The molecule of claim 2, wherein said alpha-helical oligopeptide moiety is of the formula (VII):
A-B-C-D-E-F (VII) wherein:
A is Asp or His; B is Ser; C is He or Leu;
D, E and F are alpha helix favoring amino acids, and wherein the second agent is a polypeptide comprising an amino acid sequence different from the amino acid sequence of the alpha-helical oligopeptide moiety.
14. The molecule of claim 2, wherein the second agent is a protein.
15. The molecule of claim 2, wherein the second agent is a fusion protein.
16. The molecule of claim 15, wherein the fusion protein comprises an epitope tag.
17. The molecule of claim 16, wherein the epitope tag is chosen from a His-tag, a flag-tag, a GST-tag, and an MBP-tag.
18. The molecule of claim 2, wherein the second agent is an antibody.
19. The molecule of claim 2, wherein the second agent is a receptor binding protein.
20. The molecule of claim 2, wherein the second agent is a hormone.
21. The molecule of claim 20, wherein the hormone is insulin.
22. The molecule of claim 2, wherein Y forms an alpha-helical structure independent ofZ.
23. The molecule of claim 22, wherein Y has an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40 or SEQ ID NO:41, or a variant thereof.
24. The molecule of claim 23, wherein when Y has an amino acid sequence set forth in SEQ ID NO.2 or SEQ ID NO.40 or SEQ ID NO:41, or a variant thereof, the serine corresponding to position 2 of SEQ ID NO:2 or SEQ ID NO:40 or SEQ ID NO:41, is invariant.
25. The molecule of claim 22, wherein Z is a polypeptide comprising an amino acid sequence which is different from the amino acid sequence of the alpha-helical oligopeptide moiety set forth in SEQ ID NO:2 or SEQ ID NO:40 or SEQ ID NO:41, or a variant thereof.
26. The molecule of claim 25, wherein the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40 or SEQ ID NO:41, or a variant thereof, is located at the N- terminus of the polypeptide.
27. The molecule of claim 25, wherein the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40 or SEQ ID NO.41, or a variant thereof, is located at the C- terminus of the polypeptide.
28. The molecule of claim 27, wherein the amino acid sequence set forth in SEQ TD NO:2 or SEQ ID NO:40 or SEQ ID NO:41, or a variant thereof, is located between the N-terminus and the C-terminus of the polypeptide.
29. The method of claim 1 , wherein the leaving group is 3 ' , 5 ' -ADP .
30. The method of claim 1, wherein the second agent is a polypeptide.
31. A pharmaceutical composition comprising an effective amount of a molecule of formula (I), wherein said molecule of formula (T) is:
Mq-L-Y-Z (T) wherein:
M is a first agent; q is an integer greater than 0;
L is a linking group;
Y is an alpha-helical oligopeptide moiety; and
Z is a second agent.
32. The pharmaceutical composition of claim 31 , wherein said pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
33. A method of delivering a second agent to a desired location within a subject, comprising administering to a subject a molecule of formula (I), wherein the first agent is capable of delivering the second agent to the desired location within the subject, wherein said molecule of formula (I) is:
Mq-L-Y-Z (I) wherein:
M is a first agent; q is an integer greater than 0;
L is a linking group;
Y is an alpha-helical oligopeptide moiety; and Z is a second agent.
34. The method of claim 33, wherein the second agent is a therapeutic protein.
35. A method of treating a subject, comprising adήiinistering to said subject a molecule of formula (I), such that said subject is treated, wherein said molecule of formula (I) is:
Mq-L-Y-Z (I) wherein:
M is a first agent; q is an integer greater than 0;
L is a linking group;
Y is an alpha-helical oligopeptide moiety; and Z is a second agent.
36. A method of generating a molecule of formula (VIII) :
G-Y-Z (VIII) wherein: G is a transferred moiety;
Y is an alpha-helical oligopeptide moiety; and Z is a second agent, comprising: a) providing a compound of formula (IX) :
Y-Z (IX) wherein:
Y is the alpha-helical oligopeptide moiety; and Z is the second agent; and b) contacting the compound of formula (IX) with an enzyme having phosphopantetheinyl transferase activity or a fragment thereof having phosphopantetheinyl transferase activity, in the presence of a compound of formula (X):
G-N (X) wherein:
G is the transferred moiety; and N is a leaving group; under suitable conditions to allow the enzyme or the fragment thereof to attach the transferred moiety (G) in formula (X) to the alpha-helical oligopeptide moiety (Y) in formula (IX), thereby to generate the molecule of formula (VIII).
37. The method of claim 36, wherein the transferred moiety comprises a linking group and a first agent.
38. The method of claim 36, wherein the first agent is chosen from the group consisting of a small molecule, a proton, a linker group, an affinity probe, a spectroscopic probe, a radioactive probe, a peptide, a nucleic acid, a lipid molecule, a bio-molecule, a radical generating molecule, a toxin, a polymer, a receptor binding molecule, a sugar, an antibody and a polysaccharide.
39. The method of claim 38, wherein said spectroscopic probe is selected from the group consisting of a fluorophore, a chromophore, a magnetic probe and a contrast reagent.
40. A method of generating a protein-small molecule conjugate of formula (XI):
Iq-L-Y-K (XI) wherein:
I is a small molecule moiety; q is an integer greater than 0; L is a linking group;
Y is an alpha-helical oligopeptide moiety; and K is a protein; comprising: a) providing a compound of formula (XII) :
Y-K (Xπ) wherein:
Y is the alpha-helical oligopeptide moiety; and K is the protein; and b) contacting said compound of formula (XII) with an enzyme having phosphopantetheinyl transferase activity or a fragment thereof having phosphopantetheinyl transferase activity, in the presence of a compound of formula (XIII):
Iq-L-N (XIII) wherein: I is the small molecule moiety; q is an integer greater than 0; L is the linking group; and N is a leaving group; under suitable conditions to allow the enzyme or the fragment thereof to attach the linking group (L) to the alpha-helical oligopeptide moiety (Y)5 thereby generating the protein-small molecule conjugate of formula (XI).
41. Use of a molecule of formula (I) for the treatment of a subj ect in need thereof, wherein said molecule of formula (I) is:
Mq-L-Y-Z (I) wherein:
M is a first agent; q is an integer greater than 0; L is a linking group;
Y is an alpha-helical oligopeptide moiety; and Z is a second agent.
42. A method of labeling a first protein and a second protein with a compound, said method comprising:
(a) providing a first protein and a second protein, wherein the first protein is linked to at least one alpha helical oligopeptide moiety comprising an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:40, and the second protein is linked to at least one alpha helical oligopeptide moiety comprising an amino acid sequence set forth in SEQ ID NO:41; and b) contacting said first protein and said second protein with one or more compounds in the presence of one or more phosphopantetheinyl transferase enzymes chosen from Sfp, Acps, or a fragment thereof having phosphopantetheinyl transferase activity, under conditions such that to allow the enzyme to transfer the one or more compounds to the one or more alpha-helical oligopeptide moieties in step (a).
43. The method of claim 42, wherein the first protein and the second protein are labeled simultaneously.
44. The method of claim 42, wherein the first protein and the second protein are labeled sequentially.
45. The method of claim 42, wherein the first and the second proteins are present in cell lysate.
46. The method of claim 42, wherein the first and the second proteins are present on the surface of live cells.
47. The method of claim 42, wherein the Sfp or a fragment thereof having phosphopantetheinyl transferase activity transfers a compound to an alpha-helical oligopeptide moiety having an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO.40, thereby to label the first protein.
48. The method of claim 42, wherein the Acps or a fragment thereof having phosphopantetheinyl transferase activity transfers a compound to an alpha-helical oligopeptide moiety having an amino acid sequence set forth in SEQ ID NO:41, thereby to label the second protein.
49. The method of claim 42, wherein the compound comprises the formula Iq-L-N (XIII).
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