WO2002073201A1

WO2002073201A1 - Reagents for labeling protein

Info

Publication number: WO2002073201A1
Application number: PCT/JP2002/001718
Authority: WO
Inventors: Naoto Nemoto; Toru Sasaki
Original assignee: Mitsubishi Chemical Corporation
Priority date: 2001-02-27
Filing date: 2002-02-26
Publication date: 2002-09-19
Also published as: JP2002257832A

Abstract

It is intended to provide reagents for labeling a protein which makes it possible to introduce one molecule of a labeling and one molecule of an affinity substance or a covalent bond-forming reactive group per molecule of a target protein to be labeled. Namely, reagents for labeling a protein wherein one molecule of a labeling and one molecule of an affinity substance or a covalent bond-forming reactive group are bonded to a molecule of a substance capable of binding to the C-end of a protein.

Description

Technical field for protein labeling reagents

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. Background art

Due to the progress of the Human Genome Project, the primary sequence of the entire human genome is about to be decoded in 2001. At present, research on so-called post-genome research, which is a gene interaction study, has attracted attention, and the fluorescence depolarization method (Checovich, WC , et al. (1995) Nature 375, 254-256), surface plasmon method (Jonsson U et al. (1991) Anal Biochem. 198, 268-277), fluorescence correlation spectroscopy (Eigen M. and Rigler, R (1994) Proc. Natl. Acad. Sci. USA 91, 5470-5747). & Tsu et al. Recently succeeded in developing, for the first time, a technique for observing single-molecule labeled protein molecules in water using evanescent light (Funatsu, T et al. (1995) Nature 374, 555) -559) ₀ One of the major challenges in this technology was to add one fluorescent molecule to the protein molecule.

On the other hand, 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).

If 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.

In addition, 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.

That is, according to the present invention, the following inventions are provided.

(1) One molecule of a substance capable of binding 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. reagent.

(2) a labeling substance and an affinity substance or a covalent bond-forming reactive group are respectively bound to a substance capable of binding to the C-terminus of the protein via a spacer;

The labeling reagent according to (1).

(3) General formula: X— (L ¹ ) _m -A- (L ² ) _n -Y

(In the formula, X represents a residue of a labeling substance, Υ represents a residue of an affinity substance or a reactive group capable of forming a covalent bond, and L ¹ and L ² each independently represent a divalent spacer group. , M and ぴ n each independently represent an integer of 0 or 1, and A represents a residue of a substance capable of binding to the C-terminus of the protein.

The Labe / Reich reagent according to (1) or (2), comprising a compound represented by the formula:

(4) Labeling according to any one of (1) to (3), wherein the labeling substance is a fluorescent substance

(5) 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).

(6) 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. (7) a C-terminal of the protein, comprising a step of performing protein synthesis in a transcription / translation system using a nucleic acid encoding the protein in the presence of the reagent for labeling a protein according to any one of (1) to (6); How to label.

(8) General formula: X— (L ¹ ) _m -A- (L ² ) _n -Y

(In the formula, X represents a residue of a labeling substance, Υ represents a residue of an affinity substance or a reactive group capable of forming a covalent bond, and L ¹ and L ² each independently represent a divalent spacer group. , M and ぴ n each independently represent an integer of 0 or 1, and A represents a residue of a substance capable of binding to the C-terminus of the protein)

Or a salt thereof.

(9) The compound according to (8) or a salt thereof, wherein the labeling substance is a fluorescent substance.

(10) 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 compound according to (8) or (9) or a salt thereof.

(11) 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. The compound or salt thereof according to any one of (8) to (10), which is a compound having a skeleton or an analog thereof. Brief description of the drawings

FIG. 1 shows a schematic diagram of detection of a binding protein by single-molecule imaging. In FIG. 1, in (A), a fluorescent molecule that moves randomly is not detected as a background, and in (B), light is emitted according to the residence time. In (C), 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. In FIG. 2, (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, and (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. 6 shows the results of an experiment in which Cy5-biotin-puro was immobilized on a support (streptavidin membrane or streptavidin slide glass).

FIG. 7 shows the results of quantification of a biotin-binding fluorescent molecule (DNA labeled with biotin and a fluorescent substance) by evanescent light. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, a method and an embodiment of the present invention will be described in detail.

(1) Protein labeling reagent

According to the present invention, there is provided 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. Are provided.

That is, the labelich reagent of the present invention has one molecule of a labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group in one molecule of the molecule, and has the ability to bind to the C-terminus of a protein. Having a substance.

Each of 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 as peuromycin or puromycin-like compound. Various fluorescent dyes that can be linked, for example, fluorescein series, rhodamine series, eosin series,

Anything such as an NBD sequence may be used. Specific examples of the fluorescent substance include Cy5 (Amersham), Cy3 (Amersham), IC5 (Dojindo), IC3 (Dojindo), fluorescein, tetramethylrhodamine, Texas red, and acridine orange. No. Alternatively, 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.

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 ヽ 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. -In addition, 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.

In addition, nucleosides or nucleosides and ester bonds of amino acids can also be used. Furthermore, 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. . As 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. Among these compounds, puromycin, ribocytidyl puromycin (rCpPur), deoxydilpuromycin (dCpPur), and deoxyperidyl puromycin (dU Puromycin derivatives such as pPur) are particularly preferred.

Specific examples of 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

The compound represented by these is mentioned.

The residue of a labeling substance represented by X, the residue of an affinity substance represented by Y or a covalent-forming reactive group, the residue of a substance capable of binding to the C-terminus of the protein represented by A As specific examples of the above, the residues or co- Bond-forming reactive groups.

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 type of the substituent that can be present on the carbon atom of the main chain is not particularly limited, but includes an alkyl group having 1 to 4 carbon atoms, an alkenyl group having 1 to 4 carbon atoms, an alkynyl group having 1 to 4 carbon atoms, Examples include a hydroxyl group or a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, and the like). 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. Further, the divalent spacer group may be a group derived from a polymer substance such as polyethylene and polyethylene glycol. Among the spacer groups, there are amide bonds (one CO—NH— group), ester bonds (one COO— group), thiourea bonds (one NH—CS—NH— group), and sulfone amide groups (— S 0 ₂ —NH—) and phosphodiester bond (one O—P (O) OH-0-) may be included.

m 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. A phosphoramidite, a fluorescent substance or an affinity substance, or a phosphoramidite to which a covalent bond-forming reactive group is bonded in order, followed by deprotection. 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. In addition, 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.

As a spacer for connecting a labeling substance, an affinity substance, or a reactive group capable of forming a covalent bond with a substance capable of binding to the C-terminus of a protein, an aliphatic hydrocarbon group having about 1 to 10 carbon atoms may be used. (For example, an alkylene group or an alkylene group), or a group derived from a polymer substance such as polyethylene or polyethylene daricol. As described above, 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

(In the formula, 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, and L ¹ and L ² each independently represent a divalent spacer group. , M and ぴ n each independently represent an integer of 0 or 1, and 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 Υ 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 η each independently represent an integer of 0 or 1, and Α 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 salt mentioned here includes all possible salts depending on the type of substituents present in the compound and the like, and the type of salt is not particularly limited. For example, an acid addition salt, a metal salt, an ammonium salt, or an organic amine addition salt is included. Acid addition salts include inorganic acid salts such as hydrochloride, sulfate, nitrate, and phosphate, acetate, and Organic acid salts such as oleate, fumarate, and citrate are exemplified. Examples of the metal salt 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.

In the compound of the general formula: X— (L ¹ ) _m -A- (L ² ) _n — Y, there is an isomer such as a positional isomer, a geometric isomer, a tautomer, or an optical isomer. However, all possible isomers, as well as mixtures containing two or more such isomers in any ratio, are also within the scope of the invention.

When it is desired to obtain a salt of the above compound, if the compound is obtained in the form of a salt, it may be purified as it is, or when obtained in a free form, dissolved or suspended in an appropriate solvent, The salt may be formed by adding an acid or a base to isolate and purify.

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.

(2) Method for labeling the C-terminus of a protein using a protein labeling reagent 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.

In the method for labeling the C-terminus of a protein according to the present invention, for example, in the presence of the above-mentioned labeling reagent, 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. As the coding region DNA, 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. Alternatively, 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. As the cell-free translation system, 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. When a cell-free translation system is used, 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.

In this translation system, 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. The 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 μM.

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. -When live cells are used for the translation system, 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. When a cell-free translation system is used, unreacted labeling reagent may be removed by gel filtration.

In some cases, the C-terminal labeled protein produced by the method of the present invention may be bound to a solid phase.A method for binding to the solid phase is to bind via an affinity substance or a covalent bond-forming reactive group. preferable.

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. . As used herein, the “specific polypeptide” includes a binding protein, a receptor protein constituting a receptor, an antibody, and the like.

Specific 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,

Various 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.ぴ 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).

Among these, 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. These 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.

When binding to the solid phase by means of a moiety other than an affinity substance or a covalent bond-forming reactive group, known methods that are typically used to bind proteins to the solid phase, such as tannic acid, formalin, gnoletaldehyde, The 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.

Further, in the present invention, 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.

(3) Use of the labeling reagent of the present invention

By using the labelich reagent of the present invention, 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 (in the present specification, C-terminally labeled protein) can be prepared. By utilizing such a protein, the interaction between the protein and the target molecule can be analyzed. Specifically, for example, a method including the following steps is provided.

(a) — Contact between a protein having one molecule of a first labeling substance and one molecule of an affinity substance or a covalent bond-forming reactive group in a molecule and a target molecule labeled with a second labeling substance Causing; and

(b) detecting a signal of the second label in the target molecule interacting with the protein or nucleic acid;

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. Of these, it is preferable to use a purified protein whose amino acid sequence is known, or a protein translated and purified from the cDNA library using an appropriate method.

The nucleic acid is not particularly limited as long as it has an ability to interact with the C-terminal label lig protein, and DNA or RNA can also be used. Further, the nucleic acid may have a known nucleotide sequence or function, or may have an unknown nucleic acid. Preferably, those having a known function as a nucleic acid capable of binding to a protein and having a known nucleotide sequence, or those obtained by cleavage and isolation from a genomic library or the like using a restriction enzyme or the like can be used. The sugar chain is not particularly limited, as long as it has the ability to interact with the C-terminal Labelich protein, and the sugar sequence or function may be a known sugar chain or an unknown sugar chain. Preferably, a sugar chain which has already been separated and analyzed and whose sugar sequence or function is known is used.

The low molecular weight compound is not particularly limited as long as it has the ability to interact with the c-terminal labeled protein. Even those whose functions are unknown or whose ability to bind to proteins are already known can be used.

The term “interaction” 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. Denotes the action of forces acting between molecules arising from at least one, but this term should be interpreted in the broadest sense and not in any way limited. 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. In addition, a binding reaction, a synthesis reaction, and a decomposition reaction resulting from the above actions are also included in the interaction.

Specific examples of the interaction 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 target molecule is usually used after being labeled with a labeling substance. As the labeling substance used for labeling the target molecule, use a labeling substance different from the labeling substance used for labeling the C-terminal labeled protein or the label nucleic acid.

The labeling substance is usually selected from non-radioactive labeling substances such as fluorescent substances. Examples of the fluorescent substance include various fluorescent dyes having a free functional group (for example, a carboxyl group, a hydroxyl group, an amino group, etc.) and capable of being linked to the above-mentioned target substances such as proteins and nucleic acids. For example, it may be any one of the fluorescein series, rhodamine series, eosin series, NBD series and the like. In addition, as long as the compound can be labeled, such as a dye, the type and size of the compound are not limited. Specific examples of the fluorescent substance include Cy5 (Amersham), Cy3 (Amersham), IC5 (Dojindo Chemical), IC3 (Dojindo Chemical), Honorable Resin, Tetramethylrhodamine, Texas Red, Atalidine orange and the like. Further, 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.

In the above-described analysis method, prior to the step (a), a step of quantifying the amount of the protein immobilized on the solid phase by using the signal of the first labeling substance as an index using _y . It is preferable to include In the analysis method, the process

Using the signal intensity of the second label detected in (b) and the amount of the protein immobilized on the solid phase previously quantified, the unit of the immobilized protein using evanescent light It is preferable to include a step of quantifying the strength of interaction between molecules by measuring the signal intensity per amount. In such a preferred embodiment, it becomes possible to relatively easily quantify the intermolecular interaction between the protein and the target molecule.

In such an analysis method, the method for detecting the second labeling substance labeled on the target molecule is not particularly limited, but for example, it can be detected using a fluorescence imaging analyzer.

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.

When measuring or analyzing protein-molecule interaction using this method, 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. As 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.

As a method for bringing the labeled target molecule into contact with the solid-phased molecule, any method may be used as long as the two molecules come into contact with each other to an extent sufficient to interact with each other. It is preferable to prepare a solution in which the target molecule is dissolved in a buffer solution to be used at an appropriate concentration and then contact the solution with the surface of the solid phase. After the two molecules are brought into contact with each other, a step of washing excess labeled target molecules, preferably with the same buffer, is performed, and a signal (fluorescent signal) emitted from the target molecule labeled substance remaining on the solid phase is used. ) Can be measured or analyzed using a commercially available imaging analyzer to identify molecules that interact with the immobilized molecule. In this method, for example, 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.

When 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. Specifically, when the target molecule in which the interaction is recognized is a protein, the primary structure can be identified by analyzing the amino acid sequence using an amino acid analyzer or the like. When 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.

Clarification of Japanese Patent Application No. 2000-1500, which is the application on which the priority claim of the present application is based All contents disclosed in the specification are 'disclosed herein by reference.'

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples. Example .

Example 1: Interaction analysis between kinesin molecule and microtubule molecule using single molecule labeling method and single molecule imaging method

(1) Synthesis of Cy 5-puro

Puromycin dihydrochloride (Wako Pure Chemical Industries, Ltd.) 250 mg of 30 ml of 0.3 M sodium carbonate-0.2 M sodium hydrogencarbonate-2.0 M sodium chloride aqueous solution (aqueous solution A) and 20 ml of sodium chloride After dissolving in dani methylene and stirring, the organic layer was separated. Methylene chloride (20 ml) was added to the aqueous layer, and the mixture was separated again. The organic layers were combined, concentrated with an evaporator, and then a small amount of pyridine was added and concentrated twice. It was dissolved in cetonitrile (5 ml) and pyridine (2.5 ml). Trifluoroacetic anhydride (1.0 g) was added, the mixture was stirred at room temperature for 30 minutes, cooled with ice, water (5 ml) was added, and further 20 ml of aqueous solution A was gradually added. The operation of extracting twice with methylene chloride (20 ml), concentrating the obtained organic layer, adding pyridine and concentrating the mixture was repeated twice to obtain crude -trifluoacetyl-puromycm (250 mg).

The crude α-trifluoroacetyl-puromycin (250 mg) was reacted with 1.5 equivalents of dimethoxytrityl chloride in 5 ml of pyridine for 1 hour at room temperature, separated with water and dimethylene chloride, and the organic layer was concentrated. Silica gel column chromatography (mobile layer; ethyl acetate / hexane / pyridine 90: 10: 0.5) "Purified Na-trif luoroacetyl-5'-dimethoxytrityl puromycin (Yield 360 mg) _Q

Dissolve the whole amount in 3 ml of pyridine, add 3 equivalents of succinic anhydride and 10 mg of dimethylaminoviridine (DMAP), stir at room temperature for 2 days, then separate with water and ethyl acetate and concentrate the organic layer. Then, the target product was purified through 12 g of silica gel using ethyl acetate as a mobile phase (yield: 380 mg). Succinate and 1.1 mag An amount of N-hydroxysuccinic acid was dissolved in dimethylformamide (DMF), and a DMF solution of 1.1 equivalents of hexylcarbodimide was added in an ice bath, and the mixture was stirred for 16 hours while returning to room temperature. 'The resulting precipitate was filtered off, and the filtrate was added to a DMF suspension containing NovaSyn TG. Amino resin (novabiochem) and a catalytic amount of DMAP, followed by stirring at room temperature for 16 hours. The resin was collected with a glass filter, washed with DMF, methanol, and ethyl acetate, dried with a vacuum pump, and the amino groups of the unreacted resin were acetylated by a normal capping method in DNA synthesis. Puromycin was released from a part of the obtained puromycin resin by a conventional deprotection method and quantified. The tf was calculated to be 67 μmol / gram resin.

Put the prepared puromycin resin equivalent to 10 mol into a reaction vessel for solid phase synthesis, and use the phosphoramidite method to obtain Ac-dC_CE phosphoramidite (25 equivalents), 5-amino-modified 5 (10 equivalents) (both are Darren Research) ) Were sequentially combined. Except for omitting the cap reaction, the synthesis cycle of the usual phosphoramidite method was followed, and all of the solution for fat, the solution of the activator, and the oxidation solution were those manufactured by Darren Research. After removing the monomethoxytrityl group at the 5 'end, concentrated ammonia water was added, and the mixture was left at room temperature for 6 hours for deprotection. The extracted solution was concentrated, diluted with water, neutralized by gradually adding 30% acetic acid in an ice bath, and then freeze-dried. The resulting solid was purified by reversed-phase high-performance liquid chromatography (HPLC). . Column is C0SM0SIL 0DS AR-300 (20 awake x 250 thighs)

(Nacalai) and eluted with a linear gradient of 0.1 M aqueous triethylamine solution and 80% acetonitrile-0.02 M aqueous solution of triethylamine. About 6 micromol of the desired product, aminolink-dC-puromycin, was obtained.

(MS; ESI 927.6 [M-H])

Cy5 Monofunctional Dye (Amersham Fanolemasianotech) Add 50 β1 DMF to one tube, and immediately add 80 nmol of Aminolink-dC-puromycin 0.15 M sodium carbonate buffer (pH 9.0) 100 μl 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.

(2) Kinesin DNA preparation

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 鎳 type and having a base sequence having the nucleotide sequence of 载 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

After reaction with I at 37 for 16 hours, the short fragment was removed with a Primer Remover (Edge BioSysytem Co.) and purified. On the other hand, pCITE-2a-c (+) (Clonetech Co) was replaced with restriction enzymes Nco I and Sac

Cleavage under the same conditions as in I and purification. These were subjected to a ligation reaction at 16 ° C for 1.5 hours using Ligation High (T0Y0B0. Co), and the clones were subcloned and confirmed by sequencing. -

(3) Transcription and translation

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 μl 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.

(4) Purification of Cy5-puro kinesin

Gel filtration was performed using NAP5 (Pharmacia) (equilibrated with 30 mM KC1, 80 mM PIPES, 2 mM MgCl ₂ , ImM EGTA, ImM DTT buffer) to remove free Cy5-puro in the sample.

(5) Single molecule imaging

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). diluted 1/200, enzyme systems (10mM DTT, Catarase, Glucose, Glucose Oxidase) methy丄cellcose (f inel 0. 3 ¥% ), using total internal reflection fluorescence microscope (TIRF) 2 mM ATP, under 2 mM MgCl ₂ The interaction between Kinesin and microtubules was observed directly (23-25 ° C). The images were recorded on video, and later incorporated into a personal computer with an image board. The speed of fluorescent spots moving on the microtubules and the fluorescence intensity were measured using Halcon 5.2.

(6) Interaction observation

Gel filtered samples were diluted 200 times with buffer and observed with TIRF microscope. The results are shown in Figure 3. In FIG. 3, one molecule of Cy5-purokinesin can be clearly identified as a bright spot. In the presence of 2 mM AMP-PNP, bright spots of Cy5-puro-kinesin that move in a certain direction along the microtubules were observed (Fig. 3). Example 2: Synthesis of puromycin derivative (Cy5-biotin-puro) containing fluorescent dye Cy5 and biotin and labeling of protein with it

(1) Chemical synthesis

Ac-dC-CE phosphoramidite (30 equivalents used for puromycin), biotin TEG phosphoamidite (same as 6 equivalents) to puromycin resin (equivalent to 7.5 ηιο1) and 5,5 -Amino-modified C6 (40 equivalents) (all three manufactured by Glen Research) were sequentially reacted. Deprotection with concentrated aqueous ammonia was carried out at room temperature while leaving the monomethoxytrityl group at the 5 'end, and after concentration with an evaporator, 80% acetic acid was gradually added in an ice bath for final deprotection. Purification was performed under almost the same conditions as in Example 1 to obtain amino-link-biotin-dC-puroma-isine.

MS; MALDI 150.2 [M + H] ⁺

In the same manner as in the synthesis of Cy5-Puro in Example 1, Cy5 Monofunctional Dye (Amersham Armassia Pyotech) for fluorescent labeling and purification. FIG. 4 shows the structural formula of the obtained puromycin derivative (Cy5-biotin-puro) having the fluorescent dye Cy5 and biotin.

(2) Preparation of GFP mRNA

In order to confirm whether Cy5-biotin-puro synthesized above was actually incorporated into the protein in a cell-free translation system, a green Fluorescent Protein (GFP) with a molecular weight of about 27,000 was selected as a protein and an uptake test was performed. went.

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).

Next, 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).

These 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.

(3) Label-I-Dani using cell-free translation system

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 ¾1, respectively. For 1 hour. For comparison, 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). As shown in FIG. 5, it was confirmed that labeling can be performed extremely efficiently when Cy5-biotin-puro is used, as compared with the case where Cy5-dC-Puro is used. Example 3: Cy5-biotin-puro immobilization on a support (streptavidin membrane or streptavidin slide glass)

(1) Comparison of immobilization

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 μΜ 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.

Similarly, 50 μl of Cy5-biotin-puro and 50 μl of Cy5-puro were spotted on a Streptavidin slide glass (Grainer) and allowed to stand for 1 hour. Then, after washing, fluorescence image analyzer Molecular Imager (Bio-Rad) Confirmed. Fig. 6 shows the results.

As a result, it was confirmed that Cy5-biotin-puro can bind more specifically to streptavidin membrane or streptavidin slide glass than to Cy5-puro.

For example, 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.

Therefore, by obtaining the control as described above, the protein labeled with the C5-terminal with Cy5-biotin-puro as prepared in Example 2 was immobilized on a support coated with streptavidin. Even in the case of a dani, it becomes possible to quantitatively measure the number of the immobilized proteins. Example 4: Quantification of Piotin-Binding Fluorescent Molecules (DNA Labeled with Piotin and Fluorescent Substance) by Evanescent Light

(1) Synthesis of DNA labeled with biotin and fluorescent substance

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. This was dissolved in a buffer of pH 6.8 (80 mM Pipes, 2 mM MgCl ₂ , ImM EGTA) and then annealed at 60 ° C to obtain double-stranded DNA, which was used in the following experiments.

(2) Immobilization and quantification of DNA labeled with biotin and fluorescent substance on quartz glass, and the case where the concentration of DNA labeled with biotin and fluorescent substance is low (50-300 pM) The degree of immobilization on glass was observed for each of the high concentrations (1.5 to L5nM). Biotin BSA, Streptavidin, and Oct1-conjugated DNA were flowed on quartz glass in this order, and observed with an epoxy light.

The specific method of measurement is as follows.

(Low concentration)

In each of the five figures in (1) of FIG. 7, 150 × 150 pixels (about 23 × 23 μm) 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.

(For high concentration)

-Under the condition where molecules can be observed, when the DNA labeled with biotin and fluorescent substance is more than 30 OpM, the screen becomes white, so the gauge was observed with the gauge lowered. Due to the narrow dynamic range, the concentration could be increased only up to 10 times under the same observation conditions. Nevertheless, it was observed that the screen became brighter as the density increased.

From the above measurement results, the followings were confirmed.

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 When ΟρΜ, ΙΟΟρΜ and 300 pM and DNA with high concentration of biotin and fluorescent substance are applied to the glass surface, the number of bright spots (the number of fixed molecules) is about 30, about 60, It was confirmed that it increased in proportion to about 180. On the other hand, when streptavidin was not immobilized on the glass surface as a control, no such increase was observed.

Therefore, it can be seen that the fluorescence of one molecule of TAMRA attached to DNA and labeled with biotin and a fluorescent substance can be observed. Industrial applicability

According to the present invention, there is provided 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.

Claims

The scope of the claims

1. A protein label consisting of 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. Chemical reagent.

2. The labeling reagent according to claim 1, wherein the labeling substance and the affinity substance or the covalent bond-forming reactive group are respectively bound to a substance capable of binding to the C-terminus of the protein via a spacer. .

3. —General formula: X— (L ¹ ) _m -A- (L ² ) _n -Y

(In the formula, X represents a residue of a labeling substance, Υ represents a residue of an affinity substance or a reactive group capable of forming a covalent bond, and L ¹ and L ² each independently represent a divalent spacer group. And m and ぴ n each independently represent an integer of 0 or 1, and A represents a residue of a substance capable of binding to the C-terminus of the protein)

3. The labeling reagent according to claim 1, comprising a compound represented by the formula:

4. The labeling dani test according to any one of claims 1 to 3, wherein the labeling substance is a fluorescent substance.

5. The affinity substance is a substance selected from the group consisting of biotin, maltose, guanine nucleotide, metal ion, glutathione, protein binding DNA, antigen molecule, calmodulin binding peptide, ATP, and estradiol, 5. The labeling reagent according to claim 1, wherein the covalent bond-forming reactive group is a ketone group, a dionole group, an azide group, or a psoralen.

6. A substance that has the ability to bind to the C-terminus of a protein. A compound containing any of the chemical structural skeletons of puromycin, 3,1-N-aminoacylpuromycin aminonucleoside, and 3,1-N-aminoaminosyladenosine aminonucleoside. 6. The labeling reagent according to any one of claims 1 to 5, wherein the reagent is an analog thereof.

7. A protein in a transcription / translation system using a nucleic acid encoding the protein in the presence of the protein label reagent according to any one of claims 1 to 6. A method for labeling the c-terminal of a protein, comprising a step of performing synthesis.

8. —General formula: X— (L ¹ ) _m -A- (L ² ) _n -Y

Or a salt thereof.

9. The compound or a salt thereof according to claim 8, wherein the labeling substance is a fluorescent substance.

10. The affinity substance is a substance selected from the group consisting of biotin, maltose, guanine nucleotide, metal ion, daltathione, protein-binding DNA, antigen molecule, calmodulin-binding peptide, ATP, and estradiol; 10. The compound according to claim 8 or 9, wherein the covalent bond-forming reactive group is a ketone group, a diol group, an azide group or a psoralen.

11. The substance capable of binding to the C-terminus of the protein is any of puromycin, 3,1-N-aminoacino-reviewomycin aminonucleoside, or 3,1-N-aminoaminosyladenosine aminonucleoside. 10. The compound or a salt thereof according to any one of claims 8 to 10, which is a compound having a structural skeleton or an analog thereof.