+

WO2002073201A1 - Reactifs de marquage de proteine - Google Patents

Reactifs de marquage de proteine Download PDF

Info

Publication number
WO2002073201A1
WO2002073201A1 PCT/JP2002/001718 JP0201718W WO02073201A1 WO 2002073201 A1 WO2002073201 A1 WO 2002073201A1 JP 0201718 W JP0201718 W JP 0201718W WO 02073201 A1 WO02073201 A1 WO 02073201A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
substance
labeling
group
molecule
Prior art date
Application number
PCT/JP2002/001718
Other languages
English (en)
Japanese (ja)
Inventor
Naoto Nemoto
Toru Sasaki
Original Assignee
Mitsubishi Chemical Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mitsubishi Chemical Corporation filed Critical Mitsubishi Chemical Corporation
Publication of WO2002073201A1 publication Critical patent/WO2002073201A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • C07K1/13—Labelling of peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the present invention relates to a protein labeling reagent. More specifically, the present invention relates to a protein labeling reagent comprising-one molecule of a labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group in a molecule.
  • Nemoto and colleagues discovered that adding a molecule of puromycin and a fluorescent molecule to a cell-free translation system at a certain concentration adds a fluorescent molecule specifically to the C-terminus of the synthesized protein. (Nemoto, N., et al (1999) FEBS Lett. 462, 43-46).
  • the one-molecule imaging method and the one-molecule labeling method described above can be combined, a system with unprecedented speed and quantitative properties can be constructed. Furthermore, it is technically very significant if a protein chip such as a DNA chip that enables large-scale parallel processing is successfully made. However, unlike the case of DNA, proteins have extremely difficult problems in terms of stability and purification methods. For example, those already reported as protein chips are limited to extremely stable proteins such as antibodies. At present, a method for immobilizing a protein encoded by cDNA or the like on a substrate while maintaining its function has not yet been established. Disclosure of the invention
  • the difference between the detection of DNA-DNA interaction on a so-called DNA chip and the detection of protein-protein interaction on a protein chip is the difference in the degree of protein-protein interaction (dynamic range). Means that it is necessary to detect an intensity difference of about 10 to the fourth power. It is an object of the present invention to provide a means for quickly and quantitatively measuring such an intermolecular interaction between a protein or nucleic acid and a target molecule. More specifically, the present invention relates to a protein capable of introducing one molecule of a labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group into a target protein molecule to be labeled. The task to be solved was to provide a labeling reagent.
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, puromycin, in which a fluorescent label and biotin for immobilization were bound, was attached to the C-terminal of the protein to be immobilized by using a cell-free translation system. It has been found that by specifically introducing the protein, the protein can be immobilized onto a glass membrane or the like via a streptavidin without losing its function.
  • the present inventors have found that the number of immobilized molecules can be identified very accurately by measuring the protein immobilized as described above using the single-molecule imaging method. Furthermore, the exact number of proteins immobilized as described above was identified, and the C-terminus of the protein to be examined for interaction with this protein was labeled with fluorescent molecules with different wavelengths, and the number was quantified. Above, Ebane By quantifying the number of interacting proteins by a single-molecule imaging method using light, it is possible to obtain an accurate dissociation constant ( Figures 1 and 2).
  • the present invention has been completed based on these findings.
  • the substance having an affinity of 1 to 1 is selected from the group consisting of biotin, maltose, guanine nucleotide, metal ion, glutathione, protein-binding DNA, antigen molecule, calmodulin-binding peptide, ATP, and estradiol.
  • the covalent bond forming reactive group is a ketone group, a diol group, an azide group or a psoralen;
  • the reagent according to any one of (1) to (4).
  • a substance capable of binding to the C-terminus of the protein either S, puromycin, 3,1-N-aminoacino repuromycin aminonucleoside, or 3,1-N-aminoacyl adenosine aminonucleoside.
  • the labeling reagent according to any one of (1) to (5) which is a compound having a chemical structural skeleton or an analog thereof.
  • X represents a residue of a labeling substance
  • ⁇ represents a residue of an affinity substance or a reactive group capable of forming a covalent bond
  • L 1 and L 2 each independently represent a divalent spacer group.
  • M and ⁇ n each independently represent an integer of 0 or 1
  • A represents a residue of a substance capable of binding to the C-terminus of the protein
  • the affinity substance is a substance selected from the group consisting of piotin, maltose, guanine nucleotide, metal ion, daltathione, protein-binding DNA, antigen molecule, calmodulin-binding peptide, ATP, and estradiol.
  • the covalent bond forming reactive group is a ketone group, a diol group, an azide group or a psoralen;
  • the chemical structure of the substance having the ability to bind to the C-terminus of the protein is either puromycin, 3,1-N-aminoacino repuromycin aminonucleoside, or 3,1-N-aminoacyl adenosine aminonucleoside.
  • FIG. 1 shows a schematic diagram of detection of a binding protein by single-molecule imaging.
  • a fluorescent molecule that moves randomly is not detected as a background
  • light is emitted according to the residence time.
  • the interaction between molecules can be detected by single-molecule imaging only for the C-terminal labeling method.
  • FIG. 2 shows a schematic diagram of a technique for identifying an unknown gene that interacts with a target molecule.
  • D adds a functional group or the like to the C-terminus of the target molecule in a cell-free translation system
  • E performs labeling simultaneously with protein synthesis in a cell-free translation system
  • F shows a target molecule.
  • the amount of protein immobilized in each well is quantified, and in (G), the magnitude of the interaction is measured in a few seconds in proportion to the fluorescence intensity.
  • Figure 3 shows the results of analyzing the interaction between kinesin molecules and microtubule molecules using single molecule labeling and single molecule imaging.
  • FIG. 4 shows the structural formula of a puromycin derivative (Cy5-biotin-puro) ′ having a fluorescent dye Cy5 and biotin.
  • FIG. 5 shows the results of avenolation of a protein with a puromycin derivative (Cy5-biotin-puro) having a fluorescent dye Cy5 and biotin.
  • FIG. 7 shows the results of quantification of a biotin-binding fluorescent molecule (DNA labeled with biotin and a fluorescent substance) by evanescent light.
  • a protein comprising one molecule of a substance capable of binding to the C-terminus of a protein and one molecule of a labeling substance bound to one molecule of an affinity substance or a covalent bond-forming reactive group.
  • the labeling substance and the affinity substance or the covalent bond-forming reactive group may be directly bound to the substance capable of binding to the C-terminus of the protein, or may be chemically bound via a spacer. They may be combined.
  • the labeling substance is usually selected from non-radioactive labeling substances such as fluorescent substances.
  • the fluorescent substance has a free functional group (for example, a hydroxyl group that can be converted to an active ester, a hydroxyl group that can be converted to a phosphoramidide, or an amino group), and has the above-mentioned nucleic acid derivative such asconceomycin or puromycin-like compound.
  • Various fluorescent dyes that can be linked for example, fluorescein series, rhodamine series, eosin series,
  • the fluorescent substance examples include Cy5 (Amersham), Cy3 (Amersham), IC5 (Dojindo), IC3 (Dojindo), fluorescein, tetramethylrhodamine, Texas red, and acridine orange. No.
  • a chemiluminescent substance for example, noreminol, ataridinium I, etc. may be used as the labeling substance.
  • the type of the affinity substance is not particularly limited, such as proteins, peptides, saccharides, lipids, and low molecular weight compounds as long as they have an affinity for a specific substance.
  • Specific examples of the affinity substance include biotin, maltose, guanine nucleotide, metal ion, daltathione, protein-binding DNA, antigen molecule, calmodulin-binding peptide, ATP, and estradiol.
  • Examples of the covalent bond-forming reactive group include a ketone group, a diol group, an azide group, and a psoralen.
  • nucleic acid derivative As the "substance having the ability to bind to the C-terminus of a protein" constituting the labeling reagent, a nucleic acid derivative is usually used.
  • the nucleic acid derivative is not limited as long as it is a compound capable of binding to the C-terminus of the synthesized protein when the protein is synthesized (translated) in a cell-free protein synthesis system or a living cell. However, it is possible to select one whose terminal has a similar chemical skeleton to aminoacyl tRA.
  • Representative compounds include puromycin having an amide bond, 3, -N-aminoacylbiulomycin aminonucleoside (3, -N-Aminoacylpuromycin aminonucleoside ⁇ PANS-amino acid), and tobacamine.
  • PANS-Gly with glycine in the acid part PANS-Val with palin in the amino acid part, PANS-Ala with alanine in the amino acid part, and PANS with amino acid parts corresponding to all amino acids —Amino acid dagger is fisted.
  • 3, -N-aminoaminoladenosine aminonucleoside, AA in which the amino group of 3,1-aminoadenosine and the carboxyl group of an amino acid are linked by an amide bond formed by dehydration condensation.
  • S-amino acid for example, MNS-Gly of glycine in the amino acid part, MNS-Val of palin in the amino acid part, AANS-Ala of alanine in the amino acid part, and all amino acids in the amino acid part
  • AANS-amino acid compounds corresponding to amino acids can be used.
  • nucleosides or nucleosides and ester bonds of amino acids can also be used.
  • all compounds chemically linked to a nucleic acid or a substance having a chemical structure skeleton similar to a nucleic acid and a base and a substance having a chemical structure skeleton similar to an amino acid are included in the nucleic acid derivative used in the present method.
  • the substance capable of binding to the C-terminus of protein puromycin, a compound in which a PANS-amino acid or an AANS-amino acid is bonded to a nucleoside via a phosphate group is more preferable.
  • puromycin ribocytidyl puromycin (rCpPur), deoxydilpuromycin (dCpPur), and deoxyperidyl puromycin (dU Puromycin derivatives such as pPur) are particularly preferred.
  • the substance constituting the reagent for labeling the protein of the present invention include a general formula: X— (L 1 ) m -A- (L 2 ) n -Y
  • X represents a residue of a labeling substance
  • ⁇ represents a residue of an affinity substance or a reactive group capable of forming a covalent bond
  • L 1 and L 2 each independently represent a divalent spacer group.
  • M and ⁇ n each independently represent an integer of 0 or 1
  • A represents a residue of a substance capable of binding to the C-terminus of the protein
  • Examples of the divalent spacer group represented by L 1 and L 2 include an alkylene group, an alkenylene group, an alkynylene group and a combination thereof, and one or more substituents are present on these carbon atoms. You may.
  • the number of carbon atoms in the main chain is not particularly limited, but is preferably from 1 to 10 carbon atoms, more preferably from 1 to 6 carbon atoms, and still more preferably from 1 to 4 carbon atoms.
  • the spacer Ichiki indicated L 1 an unsubstituted alkylene group, ⁇ Luque two alkylene group, an alkynylene group, or a combination thereof, more preferably unsubstituted ⁇ alkylene group, a methylene group, an ethylene group, propylene And a butylene group are particularly preferred, and an ethylene group is most preferred.
  • the divalent spacer group may be a group derived from a polymer substance such as polyethylene and polyethylene glycol.
  • n and n are preferably 1.
  • the labeling reagent is a chemical bond known per se between the above-mentioned labeling substance and the above-mentioned affinity substance or a covalent bond-forming reactive group and a substance having the ability to bind to the C-terminus of the protein via a spacer, if desired. It can be manufactured by bonding by a method. Specifically, for example, a “substance capable of binding to the C-terminus of a protein” protected with an appropriate protecting group is bound on a solid-phase carrier, and is used as a spacer using a nucleic acid synthesizer.
  • Types of above parts or types of bonding Depending on the type, they may be combined by a liquid phase synthesis method, or both may be used in combination.
  • binding of a metal ion such as nickel as an affinity substance can be carried out using a chelating reagent such as di-triaminodiacetic acid which can coordinate the metal ion.
  • an aliphatic hydrocarbon group having about 1 to 10 carbon atoms may be used.
  • an alkylene group or an alkylene group or a group derived from a polymer substance such as polyethylene or polyethylene daricol.
  • the present invention provides a method in which one molecule of a substance having the ability to bind to the C-terminus of a protein is combined with one molecule of a labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group.
  • This is related to the label reagent for proteins, and specific examples thereof include the general formula: X— (L 1 ) m —A— (L 2 ) n —Y
  • X represents a residue of a labeling substance
  • Y represents a residue of an affinity substance or a reactive group capable of forming a covalent bond
  • L 1 and L 2 each independently represent a divalent spacer group.
  • M and ⁇ n each independently represent an integer of 0 or 1
  • A represents a residue of a substance capable of binding to the C-terminus of the protein
  • the compound represented by is exemplified, but the compound is itself a novel compound and constitutes one aspect of the present invention. That is, the general formula: X— (L 1 ) m —A— (L 2 ) n -Y (where X represents a residue of a labeling substance, and ⁇ represents a residue of an affinity substance or a covalent bond forming reaction.
  • L 1 and L 2 each independently represent a divalent spacer group, m and ⁇ each independently represent an integer of 0 or 1, and ⁇ represents the C-terminal of the protein. Indicates the residue of the substance capable of binding)
  • the compound represented by or a salt thereof is also within the scope of the present invention.
  • the metal salt examples include alkaline metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, zinc salt, and the like.
  • Ammonium salt includes , Ammonium or tetramethylammonium; and the organic amine addition salts include addition salts such as morpholine and piperidine.
  • the above compounds and salts thereof may exist in the form of adducts (hydrates or solvates) with water or various solvents, but these adducts are also within the scope of the present invention. . Also, any crystal forms of the above compounds and salts thereof are within the scope of the present invention.
  • the present invention further provides a method for transcription using a protein-encoding nucleic acid in the presence of the aforementioned protein labeling reagent of the present invention.
  • the present invention relates to a method for labeling a C-terminus of a protein, comprising a step of performing protein synthesis in a translation system.
  • the protein labeled by the method of the present invention means a protein whose function is known or unknown and is used as an analysis target for interaction. Using the protein labeled by the method of the present invention, the interaction with a target molecule described below can be measured.
  • This protein may be a natural protein or a mutant thereof, or an artificial protein or a mutant thereof.
  • Natural proteins include organs and tissues of various organisms Alternatively, it also includes a library of proteins having diversity transcribed and translated from a cDNA library derived from a cell.
  • the artificial protein includes a sequence obtained by combining all or a part of the natural protein or a random amino acid sequence.
  • the coding region encoding the protein is controlled under the control of a promoter region derived from a virus or cell such as T7. And transcribe it to synthesize mRNA.
  • a sequence from which a stop codon is deleted is preferably used in order to improve the efficiency of labeling by several tens of times.
  • mRNA obtained from a living body by a method known per se can be used. These mRNAs can be prepared by expressing them in a translation system and performing protein synthesis.
  • Examples of the translation system used include a cell-free translation system and living cells.
  • the cell-free translation system or living cell is not limited as long as protein synthesis is performed by adding or introducing a nucleic acid encoding a protein into the cell-free translation system or living cell.
  • a cell-free translation system composed of an extract of a prokaryotic or eukaryotic organism, for example, an Escherichia coli, a heron reticulocyte, a wheat germ extract, or the like can be used.
  • Prokaryotic or eukaryotic organisms such as E. coli cells, can be used as the live cell translation system.
  • a cell-free translation system if the nucleic acid to be used is DNA, an RNA transcribed and synthesized by a method using a known RNA polymerase or the like is introduced as type II.
  • the C-terminus of the protein can be labeled with the labeling reagent by allowing the labeling reagent to be present at an appropriate concentration.
  • concentration of the labeling reagent to be present varies depending on the labeling reagent, nucleic acid or translation system actually used, but generally the final concentration is preferably in the range of 0.1 to 200 ⁇ M.
  • the concentration of the nucleic acid derivative to which the labeling substance and the affinity substance or the covalent bond-forming reactive group used in the present invention are bound may be, for example, although the concentration differs depending on the translation system and the like, those skilled in the art can appropriately determine the concentration by the following method or the like. That is, in the above-described system for producing a C-terminal labeling / reforming protein, a nucleic acid derivative to which a label substance is bound is added at a different concentration, and the resulting translation product is subjected to SDS polyacrylamide electrophoresis. Measure the signal intensity emitted from the label attached to the C-terminus, and select the concentration with the highest signal intensity.
  • the C-terminal labeling protein thus synthesized is lysed by a method known per se, and then gel filtration or the like (for example, Bio-Spin 6; BI0- Rad) removes unreacted labeling reagent and obtains it.
  • gel filtration or the like for example, Bio-Spin 6; BI0- Rad
  • unreacted labeling reagent may be removed by gel filtration.
  • the affinity substance or covalent bond-forming reactive group used in the present invention is a molecule that specifically binds to a specific polypeptide, and binds the specific polypeptide that binds to the molecule to the solid phase surface.
  • the “specific polypeptide” includes a binding protein, a receptor protein constituting a receptor, an antibody, and the like.
  • polypeptides / affinity substances bound to the solid phase or combinations of covalent bond-forming reactive groups include, for example, biotin-binding protein Z-biotin such as avidin and streptavidin, maltose-binding protein Z-maltose, Q tamper Protein Z guanine nucleotide, polyhistidine peptide z metal ions such as nickel or cobalt, glutathione-s-transferase / daltathione,
  • receptor proteins / ligands such as DNA binding protein ZDNA, antibody / antigen molecule (epitope), calmodulin / calmodulin binding peptide, ATP binding protein ZATP, or estradiol receptor protein / estradiol, etc. ⁇ is a ketone group z hydrazide group, diol group z hydrazide group, Azide group / alkyl group; psoralen / acid base (nucleic acid base such as pyrimidine ring or purine ring or analog thereof).
  • biotin-binding proteins such as avidin and streptavidin, maltose-binding protein / maltose, metal ions such as polyhistidine peptide / nickel or cobalt, glutathione-s-transferase Z glutathione, antibody Z antigen molecule (Epitope) and the like, and particularly preferred is a combination of streptavidinnobiotin.
  • biotin-binding proteins such as avidin and streptavidin, maltose-binding protein / maltose, metal ions such as polyhistidine peptide / nickel or cobalt, glutathione-s-transferase Z glutathione, antibody Z antigen molecule (Epitope) and the like, and particularly preferred is a combination of streptavidinnobiotin.
  • binding proteins are known per se, and the DNA encoding the protein has already been cloned.
  • the specific polypeptide described above can be bound to the solid phase surface by a method known per se. Specifically, for example, tannic acid, formalin, dartal aldehyde, pyrvic aldehyde, Benzizone diazotate, toluene-2,4-diisocyanate, an amino group, a carboxyl group, or a method utilizing a hydroxyl group or an amino group can be used.
  • reaction can be carried out using pyruvaldehyde, benzodiazobis bis-diazotide, toluene-2,4-diisocyanate, an amino group, a carboxyl group, or a hydroxyl group or an amino group.
  • a protein chip containing an aggregate of proteins can be constructed by binding a large number of the above-mentioned C-terminal labeled proteins of the present invention to a solid phase.
  • Such a protein chip is useful for analyzing a molecular interaction between a protein and a target molecule.
  • a protein having one molecule of a labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group in one molecule can be prepared.
  • the interaction between the protein and the target molecule can be analyzed. Specifically, for example, a method including the following steps is provided.
  • the target molecule mentioned above means a molecule that interacts with a C-terminal labeled protein, and specifically includes proteins, nucleic acids, sugar chains, low molecular weight compounds, and the like.
  • the protein is not particularly limited, as long as it has the ability to interact with the C-terminal Labelich protein, and may be a full-length protein or a partial peptide containing a binding active site.
  • the protein may have a known amino acid sequence and its function, or may have an unknown protein. These can be used as a target molecule even with a synthesized peptide chain, a protein purified from a living body, or translated from a cDNA library or the like using an appropriate translation system, and a purified protein or the like can be used as a target molecule.
  • the synthesized peptide chain may be a glycoprotein having a sugar chain bound thereto.
  • a purified protein whose amino acid sequence is known, or a protein translated and purified from the cDNA library using an appropriate method.
  • reaction performed by these target molecules with the c-terminal labeled protein generally means a covalent bond, a hydrophobic bond, a hydrogen bond, a Van der Waals bond, or a bond by electrostatic force between the protein or nucleic acid and the target molecule.
  • the covalent bond includes a coordinate bond and a dipole bond.
  • the coupling by electrostatic force includes not only electrostatic coupling but also electric repulsion.
  • a binding reaction, a synthesis reaction, and a decomposition reaction resulting from the above actions are also included in the interaction.
  • interaction examples include binding and dissociation between an antigen and an antibody, binding and dissociation between a protein receptor and a ligand, binding and dissociation between an adhesion molecule and a partner molecule, and binding and dissociation between an enzyme and a substrate. , Binding and dissociation between nucleic acids and proteins that bind to it, binding and dissociation between proteins in the signal transduction system, binding and dissociation between glycoproteins and proteins, or binding between sugar chains and proteins And dissociation.
  • the fluorescent substance examples include Cy5 (Amersham), Cy3 (Amersham), IC5 (Dojindo Chemical), IC3 (Dojindo Chemical), Honorable Resin, Tetramethylrhodamine, Texas Red, Atalidine orange and the like.
  • a chemiluminescent substance for example, luminol, ataridinium I, etc. may be used as the labeling substance.
  • the binding of the labeling substance to the target molecule can be carried out using an appropriate method known per se. Specifically, for example, when the target molecule is a protein, the method of forming a C-terminal as described in (1) above can be used. When the target molecule is a nucleic acid, labeling can be easily performed by a method of performing PCR using an oligo DNA primer to which a labeling substance is previously bound by a covalent bond or the like.
  • the fluorescence imaging analyzer method involves contacting a labeled molecule with a solid-phased molecule, and the interaction between the two molecules causes the fluorescence emitted from the labeled molecule remaining on the solid-phased molecule to be converted to a commercially available fluorescence. Measured using a fluorescence imaging analyzer or It is a method of analysis.
  • a C-terminal labeled protein or a labeled nucleic acid is immobilized, and a target molecule labeled with a labeling substance is brought into contact therewith.
  • a base for immobilizing a C-terminal labeled protein or labeled nucleic acid on a solid phase a dinitrocellulose membrane or a nylon membrane, which is usually used for immobilizing a protein or nucleic acid, or a plastic microplate, etc. Can be used.
  • a method of performing a large number of analyzes at the same time is a method of addressing a plurality of C-terminal labeled proteins on the solid phase surface and immobilizing the same, or a method of labeling one type of C-terminal.
  • a method of contacting a protein with a plurality of types of labeled target molecules is used.
  • the primary structure of a target molecule that has been found to interact with a C-terminal labeled protein by the above method is unknown, its primary structure can be analyzed by a method known per se if the primary structure of the target molecule is unknown. it can.
  • the primary structure can be identified by analyzing the amino acid sequence using an amino acid analyzer or the like.
  • the target molecule is a nucleic acid
  • the nucleotide sequence can be determined by a nucleotide sequence determination method using an automatic DNA sequencer or the like. Thereby, the target molecule can be identified.
  • Cy5 Monofunctional Dye (Amersham Fanolemasianotech) Add 50 ⁇ 1 DMF to one tube, and immediately add 80 nmol of Aminolink-dC-puromycin 0.15 M sodium carbonate buffer (pH 9.0) 100 ⁇ l The solution dissolved in 1 was added and left at room temperature for 1 hour with occasional stirring. Cy5-Puro was purified by reverse phase HPLC using Shim-Pack CLC-ODS (4.6 mm x 250 mm) (Shimadzu Corporation) and the solvent system described above. Label is 5, end It was presumed that the processing proceeded preferentially at the terminal amino group, and no product labeled with the amino group at the position was detected.
  • a Kinesin fragment having Nco I and Sac I restriction enzyme sites at both ends was obtained by using a pUC8 vector (phskinZ) encoding kinesin lacking the C-terminal side as a ⁇ type and having a base sequence having the nucleotide sequence of ⁇ in SEQ ID NO: 1.
  • PCR was performed using a primer having the nucleotide sequence shown in SEQ ID NO: 2. After purifying the obtained PCR product, the restriction enzymes Nco I and Sac
  • the linearized Kinesin DNAplasmid was applied to Transription Mix (Invitrogene) to a final concentration of 10 nM and reacted at 30 ° C for 15 minutes.
  • Translation Mix (Invitrogene) 30 ⁇ l and Cy5-puro (final concentration: 30 M) are added and reacted for 1 hour.
  • the kinesin is translated by reacting with one molecule of Cy5-puro specifically at the C-terminal. (Cy5-puro-kinesin) force S obtained.
  • the translated sample was separated by SDS-PAGE using 7.5% acrylamide gel, and the band was visualized with a 530DF30 band-pass filter of Fluorlmager (BioRad Co) without staining.
  • Streptavidin was immobilized on silica glass via Biotinylated BSA, and a partially biotinylated Microtubule (extracted from pig brain and stained with TMR-SE) was bound thereto. You. In order to prevent non-specific adsorption of Cy5-puro-kinesin, coat the glass surface with lmg / ml Casein (Sigma), and filter the Kinesin sample that has been genole-filtered with NAP5 with AssayBuffer (80 mM PIPES, 2 mM MgC12, ImM EGTA).
  • FIG. 4 shows the structural formula of the obtained puromycin derivative (Cy5-biotin-puro) having the fluorescent dye Cy5 and biotin.
  • the GFP used was GFPuv4 discovered by Ito et al. (Ito, Y. et al., Biochemical and Biophysical Research Communication 264, 556-560, (1999)), and was easily excited even by a 488 nm laser beam. To check it with an image analyzer.
  • a DNA construct having a T'7 promoter region and Kozak sequence upstream of this GFP was prepared as follows.
  • a DNA (SEQ ID NO: 3) having a T'7 promoter region and a Kozak sequence is referred to as type I DNA.
  • a primer containing the sequence on the 5th side of this type II (SEQ ID NO: 4) and a primer containing a part of the complementary strand on the 3rd side and GFP on the 5th side (SEQ ID NO: 5) were used.
  • PCR was performed. The conditions of PCR were 25 cycles of 20 seconds at 95 ° C, 20 seconds at 68 ° C and 20 seconds at 72 ° C, and were performed using EX Taq polymerase (Takara).
  • PCR was performed using the plasmid encoding GFPuv4 as type I, using a primer having the nucleotide sequence of SEQ ID NO: 6 and a primer having the nucleotide sequence of SEQ ID NO: 7.
  • the PCR conditions are 30 cycles of 20 seconds at 95 ° C, 20 seconds at 68 ° C, and 30 seconds at 72 ° C, using EX Taq polymerase (Takara).
  • PCR products were purified by phenol extraction followed by ethanol precipitation using Primer Rimpar (Edge Biosystem). Equimolar amounts of each of these DNA templates were added, and PCR was carried out using a primer having the nucleotide sequence of SEQ ID NO: 4 and a primer having the nucleotide sequence of SEQ ID NO: 7.
  • PCR conditions were 30 cycles of 20 seconds at 95 ° C, 20 seconds at 68 ° C and 40 seconds at 72 ° C, and EX Taq Performed using polymerase (Takara). After phenol extraction, these PCR products were purified by ethanol precipitation using a primer remover (Edge Biosystem). The purified DNA was transcribed using RiboMAX (Promega) to obtain mRNA for translation.
  • the GFP mRNA having the T7 promoter and Cy5-biotin-puro synthesized by the above method were added to the wheat germ cell-free translation system at a final concentration of 20 M and 40 3 ⁇ 41, respectively. For 1 hour.
  • experiments were also performed using Cy5-dC-Puro instead of Cy5-biotin-puro. These reaction products were separated by 15% SDS-acrylamide electrophoresis and confirmed by a fluorescence image analyzer, Molecular Imager (Bio-Rad). The results are shown in FIG. 5 (in FIG. 5, the results obtained using Cy5-dC-Puro are denoted as Cy5-Puro).
  • FIG. 5 the results obtained using Cy5-dC-Puro are denoted as Cy5-Puro).
  • Cy5-biotin-puro synthesized in Example 2 or Cy5-puro having no biotin for comparison were each dissolved in a 2 O mM (pH 8.0) tris puffer, and 500 M, 100 M The concentrations were adjusted to ⁇ and 10.
  • Each solution 501 was spotted on streptavidin membrane (Promega) and allowed to stand for 20 minutes. Next, it was washed about 10 times with a 2 O mM (pH 8.0) tris buffer, and then confirmed with a fluorescence image analyzer, Molecular Imager (Bio-Rad).
  • Fig. 6 shows the results.
  • Cy5-biotin-puro can bind more specifically to streptavidin membrane or streptavidin slide glass than to Cy5-puro.
  • the immobilization rate of Fluorpuro and Fluor-biotin-puro on streptavidin-membrene in Fig. 6 shows that after two washes, Fluorpuro was 15% and Fluor-biotin-puro was 88% C To hot.
  • the amount of Fluor-biotin-puro fixed to the slide glass was about 3.5 times that of Fluorpuro. That is, it can be seen that the immobilization of Fluor-biotin-puro on the slide glass is a sufficiently specific binding even in consideration of non-specific adsorption.
  • Example 4 Quantification of Piotin-Binding Fluorescent Molecules (DNA Labeled with Piotin and Fluorescent Substance) by Evanescent Light
  • Octl's Pou Single-stranded DNA (SEQ ID NO: 8) containing a DNA sequence that interacts with the specific domain (Nature vol. 362 852-855, 1993) and biotinylated on the 5 and 5 sides
  • Single-stranded DNA (SEQ ID NO: 9) which has a sequence and is labeled on the 5 'side with TAMRA (5-carbox rtetramethylrhodamine), was ordered to Nippon Milling from the 3' side using a DNA synthesizer. And synthesized. Purified with a reversed-phase simple column was used.
  • 150 ⁇ 150 pixels (about 23 ⁇ 23 ⁇ m) were averaged over 60 frames (about 2 seconds). The binding of non-specific biotin and DNA labeled with a fluorescent substance was examined in the absence of Streptavidin.
  • the number of biotin and fluorescent substance-labeled DNA adsorbed on the glass surface increases with the concentration of the flowed biotin and fluorescent substance-labeled DNA. 5
  • the number of bright spots is about 30, about 60, It was confirmed that it increased in proportion to about 180.
  • streptavidin was not immobilized on the glass surface as a control, no such increase was observed.
  • a protein labeling reagent capable of introducing one molecule of a labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group into one molecule of a target protein to be labeled. It became possible to do.
  • the labeling reagent of the present invention By analyzing the intermolecular interaction with the target molecule using a protein having a C-terminal labeled using the protein, it becomes possible to measure the interaction quickly and quantitatively.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)
  • Saccharide Compounds (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

L'invention concerne des réactifs de marquage de protéine qui rendent possible l'introduction d'une molécule d'une substance de marquage et d'une molécule d'une substance d'affinité, ou d'un groupe réactif formant une liaison covalente, par molécule d'une protéine cible à marquer. Elle concerne notamment des réactifs dans lesquels une molécule d'une substance de marquage et une molécule d'une substance d'affinité, ou un groupe réactif formant une liaison covalente, sont liés à une molécule d'une substance capable de se lier au C terminal d'une protéine.
PCT/JP2002/001718 2001-02-27 2002-02-26 Reactifs de marquage de proteine WO2002073201A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2001-52840 2001-02-27
JP2001052840A JP2002257832A (ja) 2001-02-27 2001-02-27 タンパク質のラベル化試薬

Publications (1)

Publication Number Publication Date
WO2002073201A1 true WO2002073201A1 (fr) 2002-09-19

Family

ID=18913408

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2002/001718 WO2002073201A1 (fr) 2001-02-27 2002-02-26 Reactifs de marquage de proteine

Country Status (2)

Country Link
JP (1) JP2002257832A (fr)
WO (1) WO2002073201A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004051270A2 (fr) * 2002-12-05 2004-06-17 Novartis Ag Technologie de marquage faisant appel a des oligopeptides

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2004113530A1 (ja) * 2003-06-18 2006-08-03 三菱化学株式会社 ラベル化蛋白質合成用ポリヌクレオチド
CA2552520A1 (fr) * 2004-01-07 2005-07-28 The Research Foundation Of State University Of New York Polymeres a empreinte proteinique comportant des sites d'emission integres
JP2011122957A (ja) * 2009-12-11 2011-06-23 Tosoh Corp 高特異的かつ高感度なタンパク質検出方法
CN116554199B (zh) * 2023-05-12 2025-03-07 湘潭大学 基于花菁染料的靶向性谷胱甘肽荧光探针的制备和应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07198594A (ja) * 1991-06-07 1995-08-01 Ciba Corning Diagnostics Corp エバネッセント波センサ用多重出力基準装置および分析方法
WO1998016636A1 (fr) * 1996-10-17 1998-04-23 Mitsubishi Chemical Corporation Molecule permettant d'homologuer un genotype et un phenotype, et utilisation de celle-ci
JPH11322781A (ja) * 1998-05-15 1999-11-24 Mitsubishi Chemical Corp タンパク質のラベル化化合物およびその化合物を用いたタンパク質のラベル化方法
JP2000139468A (ja) * 1998-11-11 2000-05-23 Mitsubishi Chemicals Corp Ｃ末端がラベル化されたタンパク質の製造方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07198594A (ja) * 1991-06-07 1995-08-01 Ciba Corning Diagnostics Corp エバネッセント波センサ用多重出力基準装置および分析方法
WO1998016636A1 (fr) * 1996-10-17 1998-04-23 Mitsubishi Chemical Corporation Molecule permettant d'homologuer un genotype et un phenotype, et utilisation de celle-ci
JPH11322781A (ja) * 1998-05-15 1999-11-24 Mitsubishi Chemical Corp タンパク質のラベル化化合物およびその化合物を用いたタンパク質のラベル化方法
JP2000139468A (ja) * 1998-11-11 2000-05-23 Mitsubishi Chemicals Corp Ｃ末端がラベル化されたタンパク質の製造方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FUNATSU ET AL.: "Imaging of single fluorescent molecules and indivisual ATP turnovers by single myosin molecules in aqueous solution", NATURE, vol. 374, 1995, pages 555 - 559, XP000876872 *
NEMOTO ET AL.: "Fluorescence labeling of the C-terminus of proteins with a puromycin analogue in cell-free translation systems", FEBS LETTERS, vol. 462, 1999, pages 43 - 46, XP004260584 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004051270A2 (fr) * 2002-12-05 2004-06-17 Novartis Ag Technologie de marquage faisant appel a des oligopeptides
WO2004051270A3 (fr) * 2002-12-05 2004-12-29 Novartis Ag Technologie de marquage faisant appel a des oligopeptides

Also Published As

Publication number Publication date
JP2002257832A (ja) 2002-09-11

Similar Documents

Publication Publication Date Title
EP1816192B1 (fr) Lieur de construction de conjugue arnm-puromycine-proteine
JP5647113B2 (ja) 多リガンド捕捉剤ならびに関連組成物、方法およびシステム
US7150978B2 (en) Recombinant template used for producing a carboxy-terminal modified protien and a method of producing a carboxy-terminal modified protein
JP3942431B2 (ja) タンパク質−分子間相互作用解析法
JP4801445B2 (ja) 生体分子の検出方法及びそれに用いる標識色素並びに標識キット
KR20050049513A (ko) Ｏ6-알킬구아닌-ｄｎａ 알킬트랜스퍼라제를 사용한 단백질라벨링
WO2002074950A1 (fr) Procede d'analyse d'interaction intermoleculaire
JPWO2004009709A1 (ja) 非天然型標識化アミノ酸および該アミノ酸−ｔＲＮＡ結合体の作製方法
WO2002073201A1 (fr) Reactifs de marquage de proteine
JP6020865B2 (ja) 核酸リンカー
WO2007046520A1 (fr) Méthode de recherche par criblage d'une protéine en utilisant un pont puromycine supporté
JPWO2005001086A1 (ja) 固定化ｍＲＮＡ−ピューロマイシン連結体及びその用途
JP5049136B2 (ja) Ｎ末端アミノ酸が標識されたタンパク質の効率的な合成方法
JP2004053416A (ja) Ｃ末端標識タンパク質を用いるタンパク質−分子間相互作用の解析方法
JP4747292B2 (ja) 翻訳テンプレートおよびそのライブラリー、それらから合成される蛋白質および蛋白質のライブラリー、ならびにそれらを構成する要素、ならびにそれらの製造法および利用方法
US20040265921A1 (en) Intein-mediated attachment of ligands to proteins for immobilization onto a support
JPWO2005050518A1 (ja) 遺伝子および／又は蛋白質のデータベースを用いた相互作用マップの作成方法、ならびに、それを実現するためのソフトウエアおよび装置
JPH0682451A (ja) リガンドまたはレセプターの測定法
JP4679870B2 (ja) キナーゼ活性検出法
JP2002107364A (ja) ノンラベル検出用生物試薬アレイ及びその作製方法
Stindl Evaluation of ligation methods and the synthesis of a specific PNA-encoded peptide library
WO2011046990A1 (fr) Récepteurs synthétiques pour l'identification de modifications post-traductionnelles des protéines
WO2014072492A2 (fr) Colorant phosphorescent et immunodosage faisant intervenir la phosphorescence et impliquant le déplacement de peptides
JP2002286715A (ja) ペプチドアレイを用いた特定のペプチドと相互作用可能な蛋白質、脂質または核酸のノンラベル検出法
WO2004053121A1 (fr) Proteine formant un complexe avec une proteine cfos, acide nucleique codant cette proteine, et procede d'utilisation de celle-ci

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载