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WO1990005785A1 - Procede d'incorporation sur un site specifique, d'aminoacides non naturels dans des proteines - Google Patents

Procede d'incorporation sur un site specifique, d'aminoacides non naturels dans des proteines Download PDF

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Publication number
WO1990005785A1
WO1990005785A1 PCT/US1989/005256 US8905256W WO9005785A1 WO 1990005785 A1 WO1990005785 A1 WO 1990005785A1 US 8905256 W US8905256 W US 8905256W WO 9005785 A1 WO9005785 A1 WO 9005785A1
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trna
protein
amino acid
aminoacyl
molecule
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PCT/US1989/005256
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English (en)
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Peter Schultz
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The Regents Of The University Of California
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Priority to AU47412/90A priority Critical patent/AU649217B2/en
Publication of WO1990005785A1 publication Critical patent/WO1990005785A1/fr
Priority to KR1019900701546A priority patent/KR900702044A/ko

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/86—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • This invention relates generally to protein biochemistry and, more particularly, to site specific modification of proteins generally useful for controlling specificity and activity of enzymes, and for altering the natural structural properties of proteins.
  • biochemists have modified protein function by either chemically altering isolated proteins or by selecting naturally occurring variants.
  • Chemical modification is typically directed to unusually reactive an solvent accessible amino acid side chains, but often the desired modification sites are not accessible or the modifications of interest are not chemically feasible. Low specificity of modification reactions dramatically hinders the usefulness of this approach.
  • naturally occurring variants are rare, usually require substantial analysis to determine the nature of any variation, and are generally limited to substitutions with naturally occurring amino acid residues.
  • novel methods are provided for site specifically incorporating an unnatural amino acid analogue into a protein, the methods comprising the steps of:
  • the protein synthesizing system is preferably an in vitro protein synthesizing system, and the preselected codon a termination codon, such as UAG (amber) , inserted at predetermined sites.
  • the unnatural amino acid analogue will typically selected from modified natural amino acids, modified uncharged amino acids, modified acidic amino acids, modifi basic amino acids, non-alpha amino acids, amino acids with altered ⁇ , ⁇ angles, and amino acids containing functional groups selected from the group of nitro, amidine, hydroxylamine, quinone, aliphatic, cyclic and unsaturated chemical groups.
  • the aminoacyl tRNA analogue the only aminoacyl tRNA molecule in the protein synthesizi system capable of recognizing the preselected codon and th preselected codon is introduced into one site of the mRNA sequence encoding the protein which typically has a molecu weight greater than about ten thousand daltons.
  • the unnatural amino acid analogue may be situated within about 100 angstroms of a substrate binding site, an enzymatic active site, a protein-protein interface, a cofactor bindi site, or a ligand (agonist or antagonist) binding site.
  • Another aspect of the present invention includes novel proteins (usually greater than about 10 Kd) that are stoichiometrically substituted at one or more predetermine sites, preferably substantially homogeneously, with an unnatural amino acid analogue.
  • Analyzing the physical or biochemical properties of the protein can determine variou properties, such as static physical properties of the polypeptide chain, mechanism of action of an enzymatic reaction, specificity of protein binding to ligand, dynami interaction of amino acid residues of a subject protein wi a substrate, folding of the protein, or interaction of the proein with other proteins, with nucleic acids or with sugars.
  • the protein is analyzed within about 10 angstroms of the unnatural amino acid analogue insertion.
  • the present invention provides methods for making multiple alternative substitutions at preselected amino acid positions of a protein comprising th steps of: a) producing one mRNA with mistranslation codon at sites in the mRNA corresponding to the preselected amino acid positions; and b) translating the mRNA in a series of two or more translation systems each comprising an aminoacyl tRNA analogue, whereby the protein produced by one translation system differs from the protein produced by another system the preselected amino acid position.
  • one amino acid position is substitute and the difference between proteins produced by the differe translation systems is predetermined by the preselection of unnatural amino acid analogues attached to the aminoacyl tRNAs.
  • the unnatural amino acid substitution may be, for example, D-phenylalanine, (S)-p-nitrophenylalanine, (S)- homophenylalanine, (S)-p-fluorophenylalanine, (S)-3-amino-2 benzylpropionic acid, or (S)-2-hydroxy-3-phenylpropionic acid.
  • Yet another aspect of the present invention relat to methods for producing an aminoacyl tRNA analogue molecul comprising the steps of: a) attaching a predetermined unnatural amino ac analogue by an aminoacyl linkage at 2' or 3 ' ribosyl hydroxyl positions on the 3 * termina nucleotide of a multi-nucleotide molecule
  • aminoacyl-multi-nucleotide molecule aminoacyl-multi-nucleotide molecule
  • tRNA(-Z) truncated tRNA molecule
  • nucleotide molecule which may be a dinucleotide such as 5'-pCpA-3 • , corresponds to a tRNA 3 ' terminus.
  • the ligation of the multi-nucleotide molecule (MNM) to the tRNA(-Z) molecule typically generates a comple tRNA molecule, and the tRNA(-Z) may be derived from a run-o transcript.
  • the attaching of the predetermined unnatural amin acid analogue by an aminoacyl linkage at 2 or 3 ' ribosyl hydroxyl positions on the 3 • terminal nucleotide of a mult nucleotide molecule is preferably accomplished by th steps of: a) protecting reactive chemical groups of the with protective agents; b) protecting reactive non-aminoacyl reactive groups of the amino acid analogue with a blocking agent; c) acylating the MNM with a blocking agen - protected amino acid analogue; and d) removing the protective agents and blocking agents from the protected reactive sites.
  • Some or all of the reactive group protecting steps are substituted with steps using blocking or protective agents selected from the group consisting of: o-nitrophenylsulfeny (NSP) ; /3-cyanoethyl (EtCNO) ; benzyl ⁇ xycarbonyl (CBZ) ; 9- fluorenylamethyloxycarbonyl (FMOC) ; 2-(4-biphenyl) isopropyloxycarbonyl (BPOC) ; vinyloxycarbonyl (VOC) ; tetrahydropyranyl (THP) ; methoxytetrahydropyranyl; and photolabile groups, including 4-methoxy-2- nitrobenzyloxycarbamates (NVOC) .
  • blocking or protective agents selected from the group consisting of: o-nitrophenylsulfeny (NSP) ; /3-cyanoethyl (EtCNO) ; benzyl ⁇ xycarbonyl (CBZ) ;
  • the protecting steps may be performed using o-nitrophenylsulfenyl (NPS) for both the blocking agents and protective agents, and the ligating of the aminoacyl-MNM to the tRNA(-Z) may be performed by the enzyme T4 RNA ligase.
  • NPS o-nitrophenylsulfenyl
  • Another aspect of the present invention comprises aminoacyl tRNA analogues having the formula X - A - Y - M, wherein:
  • Y 3 1 nucleotide sequence of a tRNA molecul such as 5 '-pCpCpA-3 • ;
  • M amino acid analogue selected from the group consisting of: i) modified uncharged natural amino acids; ii) modified acidic natural amino acids; and iii) non-alpha amino acids. These analogues will be able to direct the polymerization o the M component into a nascent polypeptide chain and can serve as an acceptor for further peptide polymerization.
  • an analogue corresponding to tRNA P c h ⁇ e, aminoacylated with (S)-p-nitrophenylalanine can be produced wherein: a) X comprises the 5 1 segment of tRNA Budapest * containing a "D loop" and part of an "anticodon loop”; b) A (anticodon) comprises the trinucleotide 5'-pCpUpA-3* ; c) Y comprises the 3' segment of tRNA c ⁇ ⁇ A containing part of ail "anticodon loop", a "variable loop", a "T ⁇ C loop", and an "acceptor stem”; and d) M is (S)-p-nitrophenylalanine.
  • the present invention further includes translatio systems comprising such aminoacyl tRNA analogues. Also included is a coupled transcription and translation system, wherein products of the transcription system are translated by a translation system comprising such aminoacyl tRNA analogues.
  • Figure 1 is a schematic representation of the method for introducing unnatural amino acids site specifically into proteins.
  • Figure 2 shows schemes for synthesizing aminoacylated pCpA.
  • FIG. 3 shows the construction of the plasmid pSG7, the vector for in vitro expression of J-lactamase.
  • Segment a is the 259-bp Ba Hl-EcoRI fragment of pKKK223-3 (Brosius and Holy, (1984) Proc. Natl. Acad. Sci. USA. 81:6929), containing the tac promoter.
  • Segment b is the 37 bp Sspl (linkered to .EcoRI-PvuI fragment of pTG2dell (Kadonaga et al., (1984) J. Biol. Chem. 259:2149) containi the first part of the gene for RTEM j ⁇ -lactamase (Sutcliffe (1978) Proc.
  • Segment c is the 1386-bp Pvul-Haell fragment of pT7-3 (Tab and Richardson, (1985) Proc. Natl. Acad. Sci. USA. 82:1074) containing the remainder of the 3-lactamase gene and the ColEl origin of replication.
  • Segment d is the 1430-bp J ⁇ ae Hael fragment of pGPl-2 (Tabor and Richardson, (1985) Proc. Natl. Acad. Sci. USA. 82:1074), containing the kanamycin resistance gene from Tn903 (Oka et al., (1981) J. Mol. Bio 147:217). This gene is oriented so as not to be under the transcriptional control of the tac promoter.
  • Segment e is the 289-bp ffaell-PvuII (ligated to the blunt-ended BamHI s of segment a to regenerate only the BamHI site) fragment f pT7-3.
  • FIG. 1 shows the jLn vitro synthesis and purification of truncated /3-lactamase.
  • Lane 1 Crude in vitro reaction
  • Lane 2 Purified 3-lacatamase synthesized vitro
  • Lane 3 Purified 0-lactamase synthesized i ⁇ _ vivo (JM101/pSG7) .
  • Figure 5 shows the tRNA PHE/CUA(-CA) sequence as determined by the enzymatic method.
  • tRNA P c h%,e A was ei.ther puri.fied by preparativ gel electrophoresis and used in chemical misacylation reactions, or treated with nucleotidyl transferase (Cudney and Guider, (1986) J. Biol. Chem.. 261:6450), gel-purifi and used in misacylation reactions with yeast PRS.
  • Figure 6 shows the test of acylated and nonacylat suppressor tRNA in vitro. Reactions (30 ⁇ L) were carried ou as described in Figure 4, cooled to 0°C and centrifuged.
  • Lanes 1, and 3 were supplemented with [ 3H]-Phe (Amersham) to a final specific activity of 190 Ci/mol; Lane 4: Reaction primed wi pF66am and 5 ⁇ g suppressor that had been acylated enzymatically with [ 3H]-Phe (speci.fi.c acti.vi.ty 9.4 Ci/mmol Phe-tRNA) .
  • Enzymatic misacylation reactions contained the following: 4 ⁇ M tRNA P c ⁇ A (30 ⁇ g, which had been desalted and lyophilized following gel purification), 80 ⁇ M phenylalanine, 40 mM Tris-HCl (pH.8.5)
  • nitrocefin hydrolysis unit (1 ⁇ mole nitrocefin hydrolyzed/min/mL, 0.1 mM nitrocefin, 50 mM phosphate buff pH 7) corresponds to 0.61 ⁇ g enzyme, as determined by Bradford assay.).
  • Figure 7 shows the method of chemical aminoacylation of the dinucleotide pCpA.
  • the dinucleotide pCpA was prepared by standard solution phase phosphotriest synthesis (Jones et al., (1980) Tetrahedron. 36:3015; Van Boom and Wreesman in "Oligonucleotide Synthesis", Gait (Ed. IRL Press, Washington, 1984) .
  • the fully protected molecule was 4-chlorophenyl-4-N-anisoyl-2*-0-tetrahydropyranyl-5'-0- [3-cyanoethyloxyphosphoryl] cytidylyl (3 '-5 1 )-[6-N, 6-N, 2 1 0, 3 '-0-tetrabenzoly] adenosine. Then o-Nitrophenylsulfeny chloride (1.8 mmol) and triethylamine (1.8 mmol) were added over six hours to pCpA (285 ⁇ mol) dissolved in dimethylsulfoxide (68 mL) .
  • 0-lactamase was purifie from 900 ⁇ L pF66am-primed reaction that had been supplement with 150 ⁇ g chemically acylated Phe-tRNA CUA following the procedure described in Figure 4. Typical yields were 0.3-0 ⁇ g (7-15%) of purified enzyme, starting from 4.5 ⁇ g in crud reaction.
  • Figure 9 shows tryptic peptide mapping of trypti digest and peptide mapping of wild-type and suppressed ⁇ - lactamase.
  • Wild-type /S-lactamase was uniformly labelled wi [ 3H]-phenylalan ⁇ .ne by m. vi.tro protein synthesis from pSG7 the presence of added [ 3H]-phenylalanme.
  • Non-labelled ⁇ - lactamase was added to the products of the in vitro synthes prior to purification of the enzyme by gel filtration on sephadex G-75 (Pharmacia) and chromatofocusing chromatograp as described in Figure 4.
  • Figure 10 shows the characterization of native an mutant 3-lactamase.
  • Wild-type and Phe 66-suppressed ⁇ - lactamase were purified to homogeneity from lmL in vitro reactions primed with pSG7 and pF66am/Phe-tRNA CUA , respectively.
  • Initial rates of nitrocefin hydrolysis were determined, at 24°C in 50 mM sodium phosphate, pH 7/0.5% DMSO, at substrate concentrations ranging from 25-250 ⁇ M. and V values were obtained from Eadie-Hofstee plots, and Bradford assay quantitations of the enzymes were used to determine k t values.
  • Kinetic parameters for the mutant enzymes were determined as follows: In vitro reactions (60 ⁇ l) containin
  • the present invention provides novel methods for synthesizing proteins containing unnatural amino acids at specific sites.
  • the methods preferably utilize modified aminoacyl tRNA's capable of polymerizing the desired unnatural amino acid(s) at unique codon(s) within an mRNA sequence. Utilizing these methods, a wide variety of unnatural amino acids may be selectively introduced into proteins of interest.
  • the methods can provide proteins whi are substantially homogeneously substituted at selected sit in stoichiometric amounts.
  • the procedures are inherently simple and allow control of both the type and site of modifications to a protein molecule within a virtually limitless number of possible variations.
  • One aspect of the invention relates to the production of modified tRNA molecules and their use in producing desired proteins as follows: a) preparing a nucleic acid sequence capable o being translated into a desired polypeptide, the nucleic a sequence including at least one codon which will be dedica to a desired preselected amino acid substitution within th polypeptide; b) obtaining or synthesizing an aminoacyl tRNA analogue which will recognize the dedicated codon and function as an adaptor molecule to direct the polymerizatio of the amino acid substitution into the polypeptide; c) combining the nucleic acid sequence with a protein translation system containing the aminoacyl tRNA analogue, whereby the translation system will function to normally translate the nucleic acid message, except that th aminoacyl tRNA analogue will direct the incorporation of th amino acid substitution for the otherwise naturally occurri corresponding natural amino acid; and d) allowing the translation system to function the sequence will be translated and the system will substitute at the direction of the selected codon the
  • Proteins are fundamental building blocks of livin organisms and serve multiple functions. Typically they ser structural functions, catalytic (or enzymatic) functions or mixture of the two. Proteins are synthesized on ribosomes which polymerize polypeptide chains out of a set of 20 comm amino acid monomers according to the information contained the sequence of nucleotides making up the "messenger RNA" (mRNA) .
  • mRNA messenger RNA
  • the mRNA is "translated” by the ribosomes which "read” three-nucleotide segments (one codon) at a time. Fr a particular AUG, or initiation codon, the ribosomes read successively in three-nucleotide segments, establishing the "frame" of translation.
  • RNA is composed of 4 different types of nucleotid containing the adenine, cytosine, guanine and uracil.
  • three-nucleotide segments codons
  • these three codons UAG, UAA and UGA, are the normal termination codons.
  • the other 61 codons have corresponding adaptor molecules (tRNA's) which recognize (o match) the message codon by co plementarily matching with these bases.
  • the complementary sequence is contained in th "anticodon" of the tRNA.
  • Another segment of this tRNA adapter molecule serves to position an amino acid at the correct site in the ribosome to serve as a substrate for th "elongation" reaction, whereby the nascent chain is polymerized to the aminoacyl moiety on the tRNA.
  • the 5' terminal codon codes for the amino terminal amino acid, and successive codons direct the successive carboxy addition of the next amino acid in the nascent chain.
  • the polypeptide chain is synthesized beginning at the amino terminus, with each subsequent amino acid added at the carboxy terminus.
  • the tRNA is enzymatically "charged” wit the correct amino acid moiety with extremely high fidelity that the adaptor molecule has the correct amino acid attach which properly corresponds to the anticodon. If a tRNA is "mischarged", that tRNA will properly recognize the anticod and the properly positioned; but improperly acylated amino acid moiety will, nevertheless, be polymerized into the nascent polypeptide chain. Furthermore, this can be extend to a tRNA which has a modified anticodon matching a termination codon. These are known as “suppressor" tRNA's, because they suppress the effect of in-frame chain termination codons which may have been introduced into a message. This phenomenon is, in part, a fundamental basis this invention.
  • This invention uses processes and molecules whic in many cases, have not been uniquely defined chemically, uses general terms which do not necessarily match the uses precisely the same by some in the field.
  • the following definitions are primarily based on functionalities.
  • Much o the state of the art and concepts utilized here are contain in Watson et al., (1987) Molecular Biology of the Gene, Vol 1 and 2, hereafter referred to as Watson et al.. Gene, specifically herein incorporated by reference.
  • reading is the process by which the translation system recognizes a given codon of the message and polymerizes, at the direction of that codon, a particul amino acid into the corresponding position of the nascent polypeptide chain.
  • misreading is used to refer to mistranslation relative to the code of correspondence betwe the codon and the amino acid inserted into the nascent polypeptide chain synthesized by the original translation system (i.e., before the selected aminoacyl tRNA is otherwi incorporated into the translation system) .
  • terminal codon refers to the codons normally used to signal translation termination in the translation system of use. Where a natural source for the translation system is used, these will typically be the codons utilized in the "universal code". Normally these ar the codons UGA, UAG and UAA. However, it is possible to generate translation systems with an entirely different correspondence of codon with amino acid, and so the term is also extended to include whatever codon is used in the syst being utilized.
  • tRNA analogue refers to any molecule which is an analogue of a tRNA with respect to the activiti of nascent peptide chain translocation and codon recognitio
  • tRNA species is not a definitive homogeneou chemical entity, since numerous methylation or other modifications or changes in the primary nucleic acid sequen may be made which may have minor or no effect on its essential properties.
  • the term is here broadened beyond it use to indicate a nebulously defined core chemical entity, including various modified forms thereof.
  • a tRNA all tRNA molecules which function to recognize a specific codon and are transcribed directly from a single gene will be considered collectively as "a tRNA".
  • the functiona definition is more relevant than a chemical description.
  • tRNAs from various sources have been defined in a general sense according to the "core" nucleoti backbone sequence (Sprinzel et al., (1987) Nucleic Acid Research 15:R53; GenBank'VIMB.L DataBank) , the number and sites of methylations and other modifications may be heterogeneous or imprecisely defined.
  • This definition is specifically intended to include each variant of a heterogeneous or homogeneous category of molecules containi minor modifications of a known tRNA including, but not limited to, differences in the methylation or other modification patterns, differences in the nucleic acid sequence of the tRNA backbone (including substitutions, additions, deletions, and modified bases) , tRNAs from exogenous sources, and other molecules which may have relevant functions common to tRNA molecules.
  • the efficienc of the interactions between the translational components e.g., ribosome, elongation factors, tRNA's
  • the translational components e.g., ribosome, elongation factors, tRNA's
  • tRNA functionalities require that the molecule may acylated by some process, enzymatic or chemical, and that t acylated molecule have adapter molecule activity.
  • the kinetics of interactions are not especially critical but ma be important in terms of efficiency.
  • aminoacyl tRNA analogue refers to any analogue of an aminoacyl tRNA molecule which: a) functions as an adapter molecule, in such a manner that it will appropriately interact with a messengerge RNA, a ribosome, associated translation and elongation factors and the nascent polypeptide chain; and b) which contains: i) a functional anticodon entity and ii) an amino acid analogue moiety that can polymerized into the nascent polypeptid chain.
  • an aminoacyl tRNA may be defined as a molecule comprising:
  • X is the 5' segment of a tRNA, consisting o nucleotides and modified nucleotides (methylations and othe modifications on the base components) making up the "D loop and part (to the anticodon entity) of the "anticodon loop";
  • A is the anticodon segment of the tRNA; narrowly defined as the 3 nucleotides which match with the codon to translated, but may be extended to include adjacent nucleotides within 3 nucleotides of the anticodon;
  • Y is the 3' segment of a tRNA, consisting of nucleotides and modified nucleotides (methylations and modifications on the base components) making up part of the "anticodon loop” and the "variable” and “T ⁇ C” loops and acceptor stem;
  • B is the 5*-pCpCpA-3' terminus of the tRNA, normally not coded by the tRNA gene and added on by the 3 * tRNA nucleotidyltransferase;
  • M is the amino acid moiety. Note that this definition is not meant to exclude the possibility of aminoacylating a shortened tRNA molecule with 3' terminal nucleotides removed, such as tRNA(-A) , tRN (-CA) or tRNA(-CCA) . If functional, they would be equivale to a tRNA as used herein.
  • multi-nucleotide refers to a sho segment of nucleic acid, typically ribonucleic acid.
  • the term is used in the context of the preparation of an aminoacyl tRNA analogue.
  • the corresponding tRNA i.e., the deacylated form
  • Z short segment
  • a truncate tRNA(-Z) can be generated directly by recombinant DNA or chemical methods.
  • the Z segment corresponds in some sense, to the multi-nucleotide(MNM) .
  • tRNA results which is equivalent to the deacylated aminoacy tRNA analogue.
  • One method of the invention uses aminoacylated-MNM's as substrates for ligation of tRNA(-Z) molecules to form aminoacyl tRNA analogues.
  • tRNA(-Z) refers to that molecule which is ligated to the aminoacyl-multi-nucleotide to produ a functional aminoacyl tRNA analogue molecule. It is particularly intended to include the component which is a shortened form of a tRNA, typically with a few of the 3' terminal nucleotides removed.
  • the ligation of the tRNA(-Z) to the aminoacyl-multi-nucleotide (aminoacyl-MNM) generates molecule which will become (or is) a functional aminoacyl tRNA analogue.
  • Z and MNM are equivalent and, in the preferred embodiment, will be the 5'-pCpA-3' dinucleotide.
  • unnatural amino acid analogue refers a molecule that is either directly an analogue or modification of an amino acid. It would include modified natural amino acids, unnatural amino acids, analogues of amino acids and derivatives of amino acids.
  • the set of natural amino acids would include tho amino acids that are commonly used in the polymerization process performed by ribosomes. Normally, these amino aci have codons which operate to signal for polymerization int protein. Although unusual amino acids exist naturally in proteins, they are usually relatively simple modifications members of the group of twenty common amino acids. Also included would be amino acids which actually do occur in nature, but are not polymerized in their final form during the polymerization (or translation) process. These natura modifications apparently result from post-polymerization modification of the amino acid that is performed either in the nascent chain stage, or more probably, upon completion the polypeptide chain. These include the post- translationally modified amino acids such as 4- hydroxyproline, 5-hydroxylysine, cystine and others.
  • Modified natural amino acids, unnatural amino acids, analogues of amino acids and derivatives of amino acids are intended to include all functional modifications analogues of amino acids, both alpha and otherwise. This would also include modifications which involve substitutio or addition of unusual atoms, addition of side groups including cofactors or their binding sites, glycosylations and acetylations.
  • preselected codon refers to a codon which is intended to be changed and will, in some function form, be within the reading frame of the protein to be produced. Thus, if a particular sequence has more than on reading frame, the codon need only be a change intended to affect one of them.
  • site specific incorporation refers to the introduction into known sites of either a particular codon into a specific site in the reading frame of a messa or of a particular amino acid analogue into a specific sit in a polypeptide chain. It will be recognized that since there is a one to one positional correspondence between co positions and their integrated amino acid sites, the site an amino acid analogue substitution is determined by its corresponding codon position. Consequently, site specific of amino acids may be derived from determination of the cod site, and vice versa.
  • the nascent polypeptide chain is the incompleted polypeptide chain resulting from the translation of the mRN which is 5' proximate to the current codon.
  • the current codon "directs" the specificity of the next amino acid analogue which is to be polymerized in the nascent chain.
  • the nascent polypeptide chai is polymerized onto the aminoacyl moiety attached to the tR which recognizes the codon adjacent to the A site of the ribosome.
  • protein and polypeptide is intended to include the products of the system that are modified molecules substantially equivalent to a protein or polypeptide. This is meant specifically to include a prote or polypeptide, as well as both its apoprotein and holoprotein forms. Included in the definition are polypeptide molecules: a) having a modified amino acid substituted at the normal site of an amino acid (equivalent to a modified amino acid) ; or b) having an amino acid like moiety which may differ in structure or composition, including, but not limited to: i) a moiety which would have the peptidyl linkage involving an amino group off a beta, gamma, delta o other carbon atom (i.e., non-alpha amino acid); ii) a moiety which might have an atom other than a carbon or nitrogen atom along the polypeptide backbone; iii) an amino acid containing a side chain R which may correspond to a synthetic side chain (including heteroatoms, cyclic or acyclic groups or metal
  • catalytic proteins include but not limited to peptidases, nucleases, glycosidases, mo and dioxygenases, pyridoxalphosphate and flavin dependent enzymes, lipases and aldolases.
  • receptor protei include antibodies, T-cell receptors, muscarinic receptors, G-proteins, lectins, DNA binding proteins and cytochromes.
  • structural proteins are included, but not limited to myosin and silk.
  • substrate binding site refers to tho the portions of the polypeptide chain whose amino acids ar located near to (or are important in conferring) the nativ three-dimensional spatial conformation of the protein important in substrate binding, or those amino acids situa nearby in space to the region where a substrate or ligand bound to the polypeptide backbone.
  • enzyme active site refers to those amino acid residues which are situated near to or are involved in conferring the essential spatial or chemical properties necessary for an enzyme to catalyze a reaction.
  • protein-protein interface refers to th residues nearby the region where distinct polypeptide chain interact.
  • cofactor binding site refers to those residues involved in, or nearby the site where a cofactor o ligand becomes attached or are involved in the recognition for where such might be attached.
  • nucleic acid sequences containing the selected codon at the specific site and Which will translat to create the polypeptide sequence of interest include but are not limited to use of natural sequences, modifications of natural sequences, partially or wholly synthetic sequences and combinations of various natural sequences to create hybrid new proteins. See, Maniatis; Wu and Grossman, Methods in Enzvmolo ⁇ y. Vol. 153, and Ausubel et al., (1987) Current Protocols in Molecular Biology. Vols. 1 and 2, each of which, is hereby specificall incorporated by reference.
  • DNA forms would include, but are not limited to, sequences integrated into a genome, sequences integrated in extrachromosomal elements (including plasmids, episomes and minichromosomes or other free DNAs) , phages, viruses, and other similar forms. Similar RNA forms are also included.
  • the sequence of the translated RNA may be changed by substituting different redundant codons at various sites. It is not well understood why one.specific codon is used instead of another redundant one, arid each redundant codon might be replaced with one of them. In theory, in the absence of "wobble", one could generate a translation syste which would utilize as many as 63 different amino acids plu one termination codon (see. Watson et al. , Gene) . By application of these techniques, one could generate a translation system with a genetic code quite different from the "universal code". In particular, the starting sequence for the desired product may be natural, a modified sequence or a totally synthetic one.
  • the site of the substitution may be changed to an codon which is intended to be generally "dedicated” to insertion of the specified preselected amino acid.
  • any codon could theoretically be chosen to code fo the mistranslation, one would normally select a codon whic is not utilized in the reading frame anywhere else in the polypeptide, and would not be translated with any existing aminoacyl tRNA contained in the ultimate translation syste to be used.
  • the termination codons are wel suited because there will not be other in frame termination codons in the sequence. Optimally a termination codon different from that actually used to terminate translation would be selected.
  • the unique codon selected may be unique by virtue of having been made so by gene synthesis. Uniqueness would result from substituting all other sites containing that codon to different redundant codons, thus leaving that particular site as the sole site containing the selected codon.
  • this system could be easily used to made two or more substitutions, of the same predetermined amino acid analogue, or of two or mo different analogues, by virtue of selecting multiple unique codons.
  • the term "substantially homogeneous” relates to t concept of homogeneity of modifications with respect to bot site and type. A particular modification is substantially homogeneous when a large majority of the resulting translation product is homogeneous, typically greater than 60% are of a single form, preferably greater than 80% identical, and optimally virtually all, greater than 98%, a identical.
  • substantially stoichiometric refers to the property that most of the products are substituted at a intended site, typically more than about 70 to 80% of the products are substituted, preferably more than 90% are substituted, and optimally virtually all, more than 98%, ar substituted.
  • a “protein synthesizing system” is a system which comprises ribosomes, tRNAs, elongation factors and all of t other components necessary to translate a mRNA into protein upon providing the mRNA and appropriate conditions. It is also referred to as a "translation system".
  • a cell inherently possesses a protein synthesizing system, bu which may have a low level of activity for various reasons. While in vivo systems may be utilized, for the uses describ herein, difficulties associated with the introduction of necessary aminoacylated tRNA analogues may exist. This may be achieved by standard microinjection procedures or by any other mechanism of introducing externally produced molecule into the cell, such as electroporation of spheroplasts. An obvious possible technique is either cell or liposome fusions, using such procedures as polyethylene glycol or Sendai viral fusions.
  • One preferred translation system is the frog oocy with microinjection, which will also find use for translati systems in other large cells. More typically, an in vitro system is preferred because it is easier to introduce a higher concentration of charged unnatural aminoacyl tRNA molecules to the system. Such systems are available commercially and have been derived from lysates of cells fr E. coli. S. cerevisiae. wheat germ and rabbit reticulocytes Inherent in the procedure is the capability for using the same single message to direct different translati systems which incorporate distinct unusual aminoacyl tRNAs. Different translation systems may be utilized to incorporat a different unusual amino acid into the selected site, thus generating a series of products, each differing by the insertion of the appropriate preselected unusual amino acid at the selected site.
  • Synthesis of a functional unusual aminoacyl tRNA involves a complicated process of: a) selection of the correct anticodon to use; a b) attachment of the appropriate predetermined amino acid analogue.
  • a tRNA with the corresponding anticodon must either be selected or manufactured. Selection is preferably performed by isolati a natural tRNA. Manufacture may be by mutation and selection, or by site specifically introducing the appropriate anticodon.
  • the modified translation system normally will not utilize the enzymatic acylation of the unusual adapter molecule, thus the functional definition of tRNA need not normally include the enzymatic acylation function. This, however, does not preclude the use of enzymatic acylation where possible, in which case an acylation capability would be important.
  • the attachment of the amino acid to a tRNA is naturally catalyzed by the aminoacyl tRNA synthetases.
  • the enzymes are reversible and are highly specific both for the appropriate tRNA (though acylation specificity does not use the anticodon for recognition) and for the amino acid to be attached. Although it may occasionally be possible to use the natural synthetases, or perhaps to modify their specificity, such will be unusual. Thus, the synthesis of the appropriate aminoacyl tRNA is very important.
  • aminoacyl tRNA is of central importance to th invention and the synthesis of the molecule is a major aspect. Where no synthetase exists for an unusual amino acid, some means must be devised to make the adapter molecule.
  • the unusual amino acid might be a substrate for a synthetase and be charged onto an appropria tRNA.
  • An alternative method for the synthesis of an unusual aminoacyl tRNA is to synthesize an aminoacyl nucleotide, and then to ligate this moiety onto the appropriate tRNA(-Z) molecule.
  • This method is generally applicable for virtually any aminoacyl tRNA molecule, including attaching normal amino acids, though much less efficient than the synthetase reactions. The only restrain are that the unusual amino acid not interfere with the acylation or deprotection steps and that it not interfere with the ligation step. If so, there is likely to be alternative chemistry to synthesize the adapter molecule.
  • the general scheme is to attach the unusual amino acid onto an oligonucleotide and then to ligate together the nucleoti portions, preferably with T4 RNA ligase.
  • a dinucleotide is preferred because it minimizes interference in the chemistr linking the amino acid to the nucleotides and provides a higher efficiency of ligation than a single nucleotide or AppA analogue.
  • the 3' terminal nucleotides on a tRNA are 5'-pCpCpA-3' , so the dinucleotide of choice is 5*- pCpA-3• .
  • nucleotides either di- or oligo
  • deoxy-C-ribo-A i.e., pdCpA
  • deoxy-RNA i.e., DNA
  • the tRNA(-Z) is ligated to the aminoacyl-multi-nucleotide (aa- MNM) to generate the final aminoacyl tRNA analogue.
  • the ligation step is performed by chemistry or by enzymatic means, the enzyme may be any which has ligation activity on single stranded RNA molecules.
  • the dinucleoti is a sufficiently long substrate for the T4 RNA ligase use in the examples, other enzymes might require a longer or shorter substrate. It will also be observed that the deprotection reactions might, in some cases, be performed after the ligation step.
  • the source of the tRNA(-Z) component may come fr processed natural tRNAs.
  • One source is gene synthesis of where the change of the anticodon of natural a termination suppressor destroys recognition o the tRNA by the aminoacyl tRNA synthetases.
  • suppressor tRNA(-Z) 's by "runoff transcription", which will not be modified as normal tRNAs, but having some substantial fraction of activity in translation. Since each tRNA molecule will typically be acylated chemically only once ( opposed to the normal enzymatic reaction) , it is generally preferable to create an aminoacyl tRNA that may function somewhat less efficiently in the elongation reaction, if v large quantities of the appropriate tRNA(-Z) can be made f convenient performance of the acylation chemistry. Use of specially designed systems for transcribing the appropriat tRNA acceptor molecules at high efficiency, but not modifi may be very important and are included as possible sources these molecules.
  • unmodified tRNA molecules are included in the specifications even though not included in the normal definition of tRNAs.
  • the chemical procedure of making the aminoacyl tRNAs may be easily modified from the described method.
  • T most obvious is to use a slightly modified tRNA(-Z) , which may be slightly longer or shorter or modified from the starting molecule. These molecules are hereby included expressly in the specifications.
  • Another obvious modification is to use, instead of a dinucleotide, a mononucleotide, trinucleotide, or other oligonucleotide. These are also included in the specifications, all include in the multi-nucleotide (MNM) molecule definition.
  • MNM multi-nucleotide
  • acylation routes which be used to synthesize the aminoacyl-dinucleotides (see Figu 1) .
  • they include blocking particularly reactive groups on the dinucleotides, attachment of the amino acid t the 3' terminal ribose ring and then removal of the blockin groups.
  • the first route involves treatment of the dinucleotides with nitrophenylsulfenyl chloride (NPS-C1) to block the cytidine base group.
  • NPS-C1 nitrophenylsulfenyl chloride
  • CDI l,l'-carbonyldiimidazole
  • Treatment with thiosulfate will remove the NPS from the cytidine leaving the aminoacyl- dinucleotide.
  • a second route involves direct synthesis of a dinucleotide or treatment of the dinucleotide with 9- fluorenylmethyloxycarbonyl chloride (FMOCC1) 8-cyanoethyl chloride (EtCNOCl) and tetrahydropyranyl chloride (THPC1) , which will block the phosphoryl groups, nucleotide base and ribose 2' hydroxyl groups. Reaction with aminoacyl - 2-(4- biphenyl) isopropyloxycarboxyl in CDI will attach to the 3' hydroxyl group.
  • FMOCC1 9- fluorenylmethyloxycarbonyl chloride
  • EtCNOCl 8-cyanoethyl chloride
  • THPC1 tetrahydropyranyl chloride
  • aminoacyl-dinucleotide Treatment with 1,1,3,3 tetramethylguanidin 2-pyridinealdoxime and formic acid will remove all the blocking groups to yield the aminoacyl-dinucleotide.
  • An aminoacyl - vinyloxycarbonyl (aa-VOC) may be substituted fo the aminoacyl-BPOC.
  • a third method of synthesis involves synthesis of or treatment of the dinucleotide with benzyloxycarbonyl (CB and tetrahydropyranyl (THP) resulting in blocking of the cytidine base and the ribose 2• and 3* hydroxyl groups.
  • CB and tetrahydropyranyl THP
  • Reaction with aminoacyl - benzyloxycarbonyl in CDI will cau attachment to the ribose 2OH group.
  • Treatment with palladium and BaS0 4 in H 2 and acid will remove the blocking groups to yield the aminoacyl-dinucleotide. It has been demonstrated that the carbobenzoxy amino acids can be coupl to pC NPSpA and the NPS and CBZ groups removed m 35% overal yield.
  • a predetermined amino acid analogue to incorporate into the polypeptide will be drive by the needs of the user. Some may desire to substitute a of a number of specific modified amino acids into the site, for any of a number of different purposes. In particular, those of most interest will be those residues which may modify specificity, activity or structure of the protein t satisfy new requirements. Part of the power of this technique is the important potential to break out of the previous limitation of choices among only the natural amino acids.
  • the present invention allows substitution of virtually any L-amino acid, natural or unnatural, as well a D-amino acids.
  • residues including, but not limited to, incorporation of: a) heavy metal atoms (useful in crystallography b) cross linking agents; c) markers (such as radioactive, spectroscopic, fluorescent, magnetic, and electronic) ; d) electron acceptors or donors; e) metal chelators; f) structurally restricting residues; and g) residues with novel nucleophilicites; h) residues with altered acidities and basicite i) residues with altered geometries (such as homoserine, homocysteine or ornithine) ; and j) residues with altered hydrogen bonding properties (e.g., amidine vs. amide) .
  • Spectroscopic markers may be introduced to particular regions in the tertiary structure of a protein o complex of polypeptides. Residues may be introduced with a different pKa, or which will affect the pKa of nearby residues, with a different nucleophillicity, or which will affect the nucleophillicity of nearby residues, with electr acceptor function, with metal chelator function, with modified hydrogen bond donor or acceptor function, with altered or restricted bond torsion angles, with cofactor binding capability or with special markers for fluorescence or other detection or purification methods.
  • the invention provides the opportunity to introduce into the polypeptide chemical groups which are beyond the range of the natural amino acid residues, and to escape from many of the constraints previously imposed by nature.
  • one of the most important uses wil be the incorporation of heavy metal scattering centers into identical locations in a polypeptide, which will, upon crystallization, allow for relative ease in solving of the wave equations necessary to determine the gross three- dimensional structure of a polypeptide chain.
  • the structur of a protein is very important, and is normally the essenti property of an enzyme which confers on it the ability to perform its function. These functions will include aspects of the properties of mechanism of catalysis, specificity of substrate binding and reaction, structural features and regulatory interactions.
  • Typical in vitro translation systems are procaryote sources including E___ coli. and eucaryote sources including rabbit reticulocyte lysates, wheat germ lysates a yeast lysates and heterogenous mixed systems containing components from various sources. (See. Wu and Grossman, Methods in Enzymology. Vol. 153) .
  • Preferred translation systems include modified transcription and translation systems exhibiting greatly increased transcription by plac the gene of interest under control of an operably linked strong promoter. Alternatively, incorporation of a very active RNA polymerase would increase the message level.
  • one particular translation sys source would be preferred.
  • the yield of desired product o further processing may be dependent upon the presence or absence of activities in the translation systems.
  • Such mi include glycosylation, acetylation or other processing enzymes, or lack of proteases or other enzymes.
  • any unnatural amino acid analogue could be substituted at the position selected
  • the unnatural amino acids which may be selected are modifications of the natural amino acids, modifications of amino acids other than the natural ones, amino acids other than alpha-amino acids (i.e. beta, gamma, etc.), amino acids having a different stereospecificity (i. D- amino acids, or having a different stereospecificity at other asymmetric carbon or other atoms) , amino acids having substituted atoms or containing unusual elements and residu containing cofactor binding sites.
  • a coupled transcription and translation system is one in which the products of the transcription system are directly translated by the system without purification or isolation of the mRNA produced.
  • the system is initially ru under conditions which are optimum for transcriptional activity after which the conditions are optimized for translation of the transcripts produced.
  • the protein products of this method may have a variety of properties, such as a) homogeneity of site and type of modifications in the proteins; b) stoichmetric modification (all of the subject proteins are modified, without dilution by unmodified forms; and c) known characterization for type and position of the modification.
  • any means of characterizing a protein should be simplified a lowered background or noise from unmodified forms or heterogeneously modified forms.
  • the means for physical or biochemical analysis is as broad as the techniques availab and applied to purified proteins, see, for example. Method in Enzymology. Vols. 1-187; Lehninger, Biochemistry; Strye Biochemistry; and Creighton, The Proteins.
  • the introductio of a heavy metal scattering center in a unique and uniform site in a protein will greatly assist in the analysis of t wave pattern data to solve the wave equations necessary to determine the three dimensional protein crystal structure (Mathews, (1976) Annual Review of Physical Chemistry. 27:4 523) .
  • Phe66 which is conserved in 4 Class A /3-lactamas (Ambler, (1979) "Beta-Lactamases", Hamilton-Miller and Smit Eds., Academic Press, New York pp. 99-125), was chosen as t first target for mutagenesis since a number of L- phenylalanine analogues are easily synthesized and phenylalanine does not require additional side chain protection in the chemical aminoacylation step.
  • a 2.5 A crystal structure of the S_j_ aureus enzyme (33% homology wi the ______ coli enzyme) localizes the residue to an extended l between a buried /3-sheet and an ⁇ -helical domain containin the active site (Herzberg and Moult, (1987) Science 236:69 The structural importance of this residue was confirmed by constructing the Phe66 ⁇ Ala (pF66A) and Phe66 ⁇ Tyr (pF66 mutants (Fig. 3) , both of which yielded little activity in crude cell extracts. (All in vivo work was carried out us E. coli strain JM101 ( lacpro thi,, supE, F ' traD36, proAB, lad*! Z ⁇ M15) .
  • the truncated gene was placed under the transcriptional control of the strong hybrid tac promoter (Amann et al., (1983) Gene 25:167), as it has been demonstrated that the amount of protein synthesized in an i vitro translation system is proportional to the amount of mRNA added (Reiness and Zubay, (1973) Biochem. Biophvs. Res Comm. 53:967).
  • the truncated gene was also placed under control of the ⁇ promoter (Tabor and Richardso (1985) Proc. Natl. Acad. Sci. USA 82:1974) from bacteriopha T7 with the intent of supplementing the reaction with T7 RN polymerase, which synthesizes RNA at a rate 10 times that o the E.
  • Yields of active / 3-lactamase synthesized in this system primed with pSG7 typically ranged from 30-45 ⁇ g/mL o reaction mixture, based on the nitrocefin hydrolysis assay (see Fig. 6) .
  • the amount of overproduction in vivo that is, JM101/pSG7 vs.
  • JMlOl/pSGl is also 11-fold, based on the specific activity of crude ce extracts.
  • T7 RNA polymerase to a final concentration of 8500 units/mL
  • T7 promoter plasmid pSGl yielded levels of active enzyme that were 65-70% of the levels produced in reactions primed with pSG7.
  • I_n vitro produced / 3-lactamase was purified to homogeneity by ammonium sulfate precipitation followed by chromatofocusing and anion exchange chromatography (Fig.
  • Protein was determined to be homogeneous by SDS- polyacryla ide gel electrophoresis and had a k ca t an ⁇ - K M ⁇ - o nitrocefin identical to that of in vivo produced enzyme. suppression work was carried out using the pSG7 derivative pF66am (Fig. 3) , which carries the Phe66 ⁇ TAG mutation.
  • the suppressor tRNA used to deliver the unique amino acid to the growing peptide chain on the ribosome mus meet two criteria: it must efficiently insert the amino ac in response to the UAG message and it must be neither acylated nor deacylated by any of the E___ coli aminoacyl-tRN synthetases present in the in vitro transcription/translati system.
  • the first condition is necessary for producing quantities of protein that can be purified and further studied, the second condition is required to insure that on the desired unnatural amino acid and not one or more of the twenty natural amino acids in the in vitro reaction will be inserted into the protein (Schimmel and Soil (1979) Ann. Re Biochem.
  • yeast tRNA P consult %,e A was effi.cient m translating UAG codons in a mammalian protein synthesizing system (although being somewhat less efficient in a wheat germ system) .
  • Kwok and coworkers Karl and Wong, (1980) Ca
  • Yeast tRNA c hue A was prepared m. mi.lli.gram quantities according to the anticodon-loop replacement procedure of Bruce and Uhlenbeck (Bruce and Uhlenbeck, (198
  • CpUpApA which includes the anticodon sequence required for amber suppressor tRNA.
  • the bands were stained with 0.02% toluidine blue, cut out and eluted witii 2 x lOmL lOOmM NaOAc (pH 4.5), ImM EDTA and 0.1% SDS.
  • the stain was removed by extractions with phenol and CHC1 3 , the tRNA half-molecules were recovered by ethanol precipitation.
  • concentration of the RNase A was increased to 2 ⁇ g/mL, and following ethanol precipitation, the pellet was resuspended in sterile water and extracted with phenol, phenol:CHC1 3 (1:1), CHC1 3 and reprecipitated with ethanol.
  • the ribonucleotide tetramer CpUpApA was synthesized by sequenti phosphotriester coupling of protected nucleosides (Jones e al., (1980) Tetrahedron 36:3015; Boom and Wreesman, (1984) "Oligonucleotide Synthesis", Gait Ed., IRL Press, Washington) .
  • the fully deprotected CUAA was ligated onto 3 » tRNA half-molecule using T4 RNA ligase supplied by Taka Shuzo.
  • the conditions for the ligation were 50 ⁇ M ATP, 19 tRNA (both half-molecule s are present in the reaction) , 8 ⁇ M CUAA and 50 U/mL T4 RNA ligase.
  • Ligation reactions were typically carried out on 5 mg of the RNase A-digested tRNA a reaction volume of 10 mL.
  • the kinase treatment was carr out with 4 ⁇ M tRNA, 120 ⁇ M ATP and 50 U/mL of T4 polynucleotide kinase (Richardson (1965) Proc. Natl. Acad. Sci. USA 54:158; and Midgley and Murray (1985) EMBO J. 4:2695).
  • the final ligation was done with 25 U/mL of T4 R ligase.
  • the suppressor produced by this method is missin the 3' terminal pCpA aminoacyl acceptor stem.
  • These nucleotides can be replaced using the tRNA repair enzyme nucleotidyl transferase (Cudny and Guider, (1986) J. Bi Chem. 261:6450) to yield a full-length yeast tH A P c h ⁇ e h .
  • suppressor tRNA can be aminoacylated in vitro with [ 3H]-Ph to levels of 30-35% (based on radioactivity incorporated i purified [ 3 H]-Phe - tRNA P c u ⁇ A ) using a large excess of ye
  • Enzymatic misacylation reactions 300 ⁇ L total volum contained the following: 4 ⁇ M tRNA c ⁇ e A (30 ⁇ g, which had been desalted and lyophilized following gel purification) , ⁇ M phenylalanine, 40 mM Tris-HCl (pH 8.5), 15 mM MgCl 2 , 45 ⁇ g/mL BSA, 3.3 mM DTT, 2 mM ATP and 22 Units yeast PRS (wh
  • 1 unit activity incorporates 100 pmol Phe in 2 minutes at 37°C under the following conditions: 2 ⁇ M tRNA Pne
  • Th tRNA was then desalted on a Pharmacia fast desalting colum and lyophilized.
  • the lyophilized mixture of acylated and non-acylated tRNA was stored at -80°C until immediately pr to its use in in vitro protein synthesis reactions.
  • wild-type yeast tRNA Phe acylates to levels of 40-45% with yeast PRS.
  • yeast tRNA P c - j A i.s not recognized b the ]_____ coli aminoacyl-tRNA synthetases present in our in vitro system (Fig. 6) .
  • Hecht and coworkers (Heckler et al., (1984) Tetrahedron 40:87; and Heckler et al., (1984) Biochemistry 23:1468) simplified this problem by chemicall acylating the dinucleotide pCpA and enzymatically ligating to the 3' terminus of a truncated tRNA [tRNA(-CA)] using T RNA ligase to afford an aminoacyl-tRNA.
  • tRNA(-CA) truncated tRNA
  • the general strategy for chemical acylation of p involves carboxyl activation of an N-blocked amino acid followed by coupling via an ester linkage to the diol of t terminal adenosine (the 2' and 3' acyl groups rapidly interconvert in aqueous solution) .
  • Aminoacylation is complicated by preferential acylation of the exocylic amin group of cytidine and 2', 3' diacylation of adenosine.
  • Th ⁇ -amino protecting group greatly increases the stability o the aminoacyl ester linkage to hydrolysis and avoids polymerization during carboxyl activation Schubert and Pinc (1974) Biochimie 56:383).
  • the protecting group mu be removed if the acylated-tRNA is to function as an A site donor. It has recently been shown by Brunner (Kreig et al. (1986) Proc. Natl. Acad. Sci. USA 83:8604; Wiedmann et al., (1987) Nature (London) 328:830; Johnson et al., (1976) Biochemistry 15:569; Baldini, et al., (1988) Biochemistry 27:7951) that ⁇ -amino protected aminoacyl pCpA can be deprotected before ligation to tRNA(-CA) without hydrolysis of the aminoacyl ester linkage.
  • NPS-C1 O-nitrophenyl sulfenyl chloride
  • NPS-pCpA was acylated with N-blocked Phe using N,N' carbonyldiimidazole as the activating agent.
  • the NPS protecting groups were removed from cytidine and the amino acid in high yield using aqueous thiosulfate (Lapidot et al (1970) Biochem. Biophys. Res. Comm.
  • Chemical acylation reactions contained the following: 600 ⁇ M pCpA-Phe (40 ⁇ g) , 10 ⁇ M tRNA Phe ⁇ cA (20 ⁇ g, which had been desalted and lyophilized following gel purification), 55 mM HEPES (pH 7.5), 250 ⁇ M AT 15mM MgCl 2 , 20mg/mL BSA, DMSO (to 10% v/v) and 200 units T4 RNA ligase. The reaction mixture was incubated at 37°C for 12 minutes, quenched by addition of 2.5 M NaOAc (pH4.5) to 10% v/v and treated as described above, but with only one round of extraction/precipitation.
  • the di-NPS protected aminoacyl pCpA was also a substrate for T4 RNA ligase, but better yields of the aminoacyl tRNA were obtained by deprotection followed by ligation rather than ligation and subsequent deprotection.
  • Fully deprotected pCpA-Phe was ligated directly t tRNA P c ⁇ e A (-CA) using T4 RNA ligase (note that the truncate suppressor tRNA is generated directly by the anticodon loop replacement method) .
  • the yield of Phe-tRNA P c y e A is 35% based on analysis of 3H-Phe incorporation into the purified suppressor (gel electrophoresis indicates 80-90% of the tRNA P c hue A (-CA) i.s converted to materi.al wi.th the same mobility of tRNA ⁇ ) .
  • tRNA 1 * ⁇ ( CA) was also aminoacylated with D-phenylalanine (D-Phe) , (S p-nitrophenylalanine (p-N0 2 ⁇ Phe) , (S)-homophenylalanine (2- amino-4-phenylbutanoic acid, HPhe), (S)-p-fluorophenylalani (p-FPhe) , (S)-3-amino-2-benzylpropionic acid (ABPA) and (S) 2-hydroxy-3-phenylpropionic acid (PLA) (in this case no ⁇ - hydroxyl protection was used).
  • aminoacyl tRNA's were used in in vitro protein synthesis to synthesize mutant ⁇ - lactamases (vide infra) .
  • Current efforts to optimize aminoacylation include the use of acid labile protecting groups and protecting groups that can be removed by hydrogenation, as well as an investigation of the use of no selective lipases for the aminoacylation of unprotected RNA Protecting groups which can be removed by hydrogenolysis or acid treatment will also simplify protection of unnatural amino acid side chains.
  • Phe72. / S-lactamase was synthesized in vitro from pSG7 in t presence of added [ H]-phenylalanine.
  • the purified radiolabelled enzyme was digested with trypsin and the fragments were separated by reversed-phase FPLC (Fig. 9) .
  • Four discrete radioactive peaks were observed, in agreement with the locations of [ 3 H]-Phe in RTEM /3-lactamase (Sutcliffe, (1978) Proc. Natl. Acad. Sci USA 75:3737; Amble and Scott (1978) Proc. Natl. Acad. Sci. USA 75:3732; Pollit and Zalkin, (1983) J. Bacteriol. 153:27; Fisher et al. , (19 Biochemistry 19:2895 and Knowles (1985) Ace. Chem. Res.
  • PLA were each loaded onto suppressor tRNA as described abov
  • In vitro protein synthesis reactions carried out in the presence of [ 35S]-methi.onme resulted m. si.mi.lar levels of radioactivity incorporated into trichloracetic acid (TCA)- precipitable material for the p-FPhe, p-N0 2 Phe and HPhe reactions.
  • TCA trichloracetic acid
  • Kinetic analyses of the ?-lactamases synthesiz in these reactions demonstrate similar M 's but different ⁇ t' s ( Figure 10). Direct quantitation of the purified p N0 2 Phe and HPhe mutants was impossible, as both mutants lo activity during purification attempts.
  • Sufficient protein can be purified to characteriz the catalytic constants and specificity of the mutants, to carry out limited mechanistic and mapping studies and to probe protein structure with techniques such as ESR and fluorescence spectroscopy. Improvements in in vitro protei synthesis, methods for tRNA generation, and tRNA aminoacylation chemistry will permit production of milligra quantities of mutant proteins via this strategy.
  • t present invention provides improved means for producing modified proteins.
  • the methods are rapid, simple and universal in utility.

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Abstract

Nouveaux procédés de production de protéines contenant des aminoacides non naturels sur des sites spécifiques. Lesdits procédés peuvent utiliser des ARNt aminoacyle modifiés capables de polymériser, au niveau de codons uniques dans une séquence d'ARNm un aminoacide non naturel désiré.
PCT/US1989/005256 1988-11-18 1989-11-15 Procede d'incorporation sur un site specifique, d'aminoacides non naturels dans des proteines WO1990005785A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU47412/90A AU649217B2 (en) 1988-11-18 1989-11-15 Method for site-specifically incorporating unnatural amino acids into proteins
KR1019900701546A KR900702044A (ko) 1988-11-18 1990-07-18 비천연 아미노산 단백질에 부위특이적으로 첨가하는 방법

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US27345588A 1988-11-18 1988-11-18
US273,455 1988-11-18
US33760189A 1989-04-13 1989-04-13
US337,601 1989-04-13

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WO1990005785A1 true WO1990005785A1 (fr) 1990-05-31

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EP (1) EP0446299A4 (fr)
JP (1) JPH04504651A (fr)
KR (1) KR900702044A (fr)
AU (1) AU649217B2 (fr)
WO (1) WO1990005785A1 (fr)

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AU4741290A (en) 1990-06-12

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