JP2005291836A - Functional tag for concentration of target substance and method of using the functional tag - Google Patents

Abstract

【Task】
Provided are a functional tag useful for detection and purification of various target substances, particularly proteins, a target substance detection method using the functional tag, and a purification method.
[Solution]
A functional tag comprising an affinity peptide tag and a reactive group Z and represented by the following formula (1);
(His) l- (X-His) m- (X) n- (Y) pZ (1)
(Wherein, X is any amino acid or amino acid derivative except histidine, Y is a linker, l is 0 or 1, m is an integer of 4 to 100, n is 0 or 1, and p is 0. Or 1).
[Selection figure] None

Description

  The present invention relates to a functional tag for concentration of a target substance and a method for using the functional tag. More specifically, the present invention relates to a functional tag useful for detection and purification of various target substances, particularly proteins, a target substance detection method using the functional tag, and a purification method.

  In order to understand life systems and further elucidate disease states, we find proteins (target proteins) that fluctuate qualitatively and quantitatively as diseases and physiological conditions change, and then investigate the functions of the proteins, It is necessary to analyze interacting proteins. Proteome analysis, which comprehensively analyzes proteins present in living tissues and cells, is attracting attention as a new approach that enables such analysis. In order to perform such proteome analysis, it is necessary to efficiently collect a minute amount of protein present in a living tissue / cell.

Conventionally, such proteins are collected by a method of recovering an expressed protein using a histidine tag (His-Tag) or a method of fixing and recovering a biotinylated protein using the affinity between avidin and biotin. Has been done in. For example,
Oligomeric purification method using a silicon-containing compound to which 2 to 3 molecules of biotin are added as a capture tag (Patent Document 1), and a labeling reagent having biotin as a marker residue and a reactive group that binds to a target substance (patent Document 2).

  The histidine tag (His-Tag) is an oligopeptide linked with histidine, which is an amino acid, and has a specific affinity for Ni2 + ions and the like. Therefore, the target protein can be purified by adding the histidine tag to the N-terminus or C-terminus of the target protein for expression, and utilizing the affinity between the histidine tag and Ni2 +. However, in order to obtain a target protein having the histidine tag, it is necessary to incorporate and express a DNA fragment encoding the tag into a gene encoding the protein, and therefore its use is limited. In addition, there has been an attempt to synthesize and use the histidine tag (Non-patent Document 1), but there is a problem that the efficiency of the reaction for peptide bonding of His and His is very low, so that the practicality is poor.

In addition, the method of biotinylating protein and immobilizing and recovering with avidin is widely used because biotin can be easily synthesized and cheap, but biotin and avidin can be used in the presence of protein denaturants and solubilizers. There was a problem that would not combine.
For this reason, it can efficiently collect minute amounts of protein present in living tissues and cells, is easy to manufacture, inexpensive, and stable even in the presence of protein denaturants, etc. An affinity peptide tag has been sought.
Special table 2001-520679 gazette JP 2004-4048 A Josper V. Olsen et al., Mol Cell Proteomics. 2004, Jan; 3 (1): 82-92.Epub, 2003

  An object of the present invention is to provide a functional tag useful for detection and purification of various target substances, particularly proteins, a method for detecting a target substance using the functional tag, and a purification method.

  In order to solve the above problems, the present inventors have conducted research. As a result, the reaction efficiency is very low when histidine is continuously linked by a peptide bond, but the reaction efficiency increases when another amino acid or an amino acid derivative is sandwiched between histidine and histidine, and histidine and another amino acid are combined. The functional tag used as the binding unit was also found to have high affinity with the chelated transition metal ion, particularly Ni2 + ion, similarly to the functional tag made of histidine. The present invention has been made based on such findings.

Accordingly, the present invention provides a functional tag comprising an affinity peptide tag, a linker Y, and a reactive group Z, wherein the affinity peptide tag has at least 3, preferably 4 or 5 or more (X-His) units. And X is any amino acid or amino acid derivative, providing the functional tag.
The present invention also relates to a functional tag comprising an affinity peptide tag and a residue Z having a reactive group, wherein the affinity peptide tag has at least 3, preferably 4 or 5 or more (X-His ) Units, and X is any amino acid or amino acid derivative.
Furthermore, the present invention provides an affinity peptide tag and a functional tag comprising a reactive group Z and represented by the following formula (1):
(His) l- (X-His) m- (X) n- (Y) pZ (1)
(Wherein, X is any amino acid or amino acid derivative except histidine, Y is a linker, l is 0 or 1, m is an integer of 4 to 100, n is 0 or 1, and p is 0. Or 1).

Furthermore, the present invention provides a protein detection kit and a protein purification kit containing the functional tag.
Furthermore, the present invention comprises mixing the functional tag with a sample containing a target substance, contacting the mixture with a carrier that holds a metal ion that binds to the affinity peptide tag, and then removing the function from the carrier. A method for detecting a target substance, comprising collecting a sex tag, is provided.
Furthermore, in the present invention, an affinity tag or a functional tag obtained by adding a detection site such as a fluorescent reagent or an RI reagent to linker Y is mixed with a sample containing a target substance, and the mixture is mixed with the affinity peptide tag. A method of detecting with high sensitivity by contacting with a carrier holding a metal ion to be bound, then recovering the functional tag from the carrier, concentrating a trace target substance present in a complex sample provide.

  Furthermore, the present invention comprises mixing the functional tag with a sample containing a protein, contacting the mixture with a carrier that holds a metal ion that binds to the affinity peptide tag, and then from the carrier to a target molecule. A protein purification method is provided, which comprises recovering the bound functional tag.

  Furthermore, the present invention comprises mixing the functional tag with a sample containing a protein, contacting the mixture with a carrier holding a metal atom that binds to the affinity peptide tag, and then from the carrier to a target molecule. Provided is a two-step protein purification method that recovers the bound functional tag and further uses an antibody in combination with a Western blotting method.

  As used herein, “affinity peptide tag” refers to a peptide tag having an affinity for a chelated transition metal ion, particularly Ni2 +. In this specification, “linker Y” refers to a portion that connects an affinity peptide tag and a reactive group, and is a compound that binds to either the N-terminus or C-terminus of the affinity peptide tag and introduces a reactive group Those capable of binding to the non-reactive group. In addition, the “residue Z having a reactive group” in the present specification refers to a functional group that selectively binds to a target substance, an atomic group that has a group that can selectively bind to a target substance, or a target substance and a selective group. A molecule that can bind. In the functional tag of the present invention, “linker Y” and / or “residue Z” may be arranged at either the N-terminus or C-terminus of the affinity peptide tag.

  The advantages of the functional tag of the present invention are that it can be artificially mass-produced, can be labeled with any stable isotope using a commercially available stable isotope-labeled F-MOC derivative amino acid, and can easily be enzyme-linked to the linker site. Digestion sites can be introduced, various functional amino acids (including non-natural amino acids) can be easily introduced, ionization efficiency during mass spectrometry is high, and amino acids between His are selected to have affinity for reversed-phase columns Therefore, there is an advantage that it can be easily separated from other substances. Here, the functional amino acid refers to, for example, an amino acid whose steric structure is changed or cleaved by light irradiation, and an amino acid having a reactive group for binding another reagent.

  Therefore, by using the functional tag of the present invention, not only proteins but also many target substances can be concentrated, purified, and detected. In addition, it is possible to efficiently detect and selectively concentrate a specific protein that is present in a very small amount in a living body. In particular, by adding detection sites such as fluorescent reagents and radioisotope reagents to the affinity peptide tag or linker Y, it is possible to concentrate a small amount of target in a complex sample and detect it with high sensitivity. Can do. Furthermore, purification of membrane proteins that require surfactants or high salt concentration buffers for stabilization has been difficult because avidin and biotin are difficult to bind, but the functional tag of the present invention should be used. Can be done more efficiently.

Next, the present invention will be described in detail based on the embodiment.
The functional tag of the present invention is a functional tag including an affinity peptide tag and a reactive group Z and represented by the following formula (1).
(His) l- (X-His) m- (X) nY (p) -Z (1)
In the formula, X is any amino acid or amino acid derivative excluding histidine, Y is a linker, l is 0 or 1, n is 0 or 1, and p is 0 or 1. In the formula, m is an integer of 4 to 100, preferably 5 to 25, and more preferably 6 to 15. Formula (1) indicates that the linker Y and / or residue Z is bound to either the C-terminus or the N-terminus of the affinity peptide tag. In the formula, “His” means a derivative of histidine having affinity for histidine or an ion such as chelated Ni.

  The target substance of the present invention is not particularly limited as long as it is a compound that can selectively bind to the reactive group of residue Z. Examples of the target substance include various biomolecules such as peptides, proteins, glycoproteins, polysaccharides, lipids, phospholipids, DNA, and RNA, and various naturally occurring or synthesized compounds. In particular, the functional tag of the present invention exhibits an excellent effect when concentrating and detecting biomolecules present in a small amount in a living body as a target substance.

  The affinity peptide tag of the present invention refers to a peptide fragment represented by (His) 1- (X-His) m- (X) n in formula (1). In the formula, X is any amino acid or amino acid derivative. The amino acid is not particularly limited as long as it includes both naturally occurring amino acids and non-natural amino acids and can be used in the affinity peptide tag of the present invention.

  Examples of the amino acids include alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan and tyrosine. In the case where the affinity between the affinity peptide tag of the present invention and the chelated Ni 2+ ion is increased, it is preferable to use cysteine or tryptophan having affinity for the ion as X. In addition, the target substance can be purified with higher purity by selecting X from a nonpolar amino acid, a polar uncharged side chain amino acid or a polar charged amino acid according to the properties of the affinity column used for purification.

The amino acid derivatives include, but are not limited to, the following compounds corresponding to the amino acids. In the formula, R is a group specific to each amino acid.
RCH (NH2) CO- [acyl group]
RCH (NH2) CO21- [anion]
RCH (NH2) CONH2 [amide]
RCH (NH2) CH2OH [alcohol]
RCH (NH2) CHO [aldehyde]
RCH (CO2H) NH- [N-Substituent]
Among these derivatives, examples of histidine derivatives include histidyl, histidinate, histidine amide, histidinol, histidinal, and N-histidino, and alanine derivatives include alanyl, alaninate, alaninamide, alaninol, alaninal and alanino. In addition, derivatives of glycine include glycyl, glycinate, glycinamide, glycinol, glycinal, and glycino.

  In addition, the reaction efficiency of a target molecule and a functional tag improves because the functional tag of this invention exists in the sample containing a target molecule in large quantities. Therefore, the ability to easily remove the unreacted functional tag is a great advantage for the functional tag. This property depends on the properties of the affinity peptide tag. It is possible to have such characteristics by selecting X of (His-X) m.

  The linker Y of the present invention is not particularly limited as long as it links the affinity peptide tag and the residue Z having a reactive group. For example, a molecule that binds to the C-terminus or N-terminus of the affinity tag and binds to a group other than the reactive group of residue Z. The linker Y preferred in the present invention is a peptide containing 3 to 30, preferably 3 to 15, more preferably 3 to 9 amino acids or amino acid derivatives. A single amino acid or amino acid derivative can also be used. The amino acid and amino acid derivative used in the linker Y may be the same as those used in the affinity peptide. In addition, by attaching the various amino acid derivatives mentioned above to the C-terminus of the linker Y, the residue Z having various reactive groups can be incorporated into the functional tag.

  Examples of the linker Y other than the peptide include a single amino acid, an amino acid derivative, a fatty acid, or a fatty acid ester, and the linker Y having a long carbon chain as a spacer if necessary. Can be introduced into sex tags. Furthermore, in the present invention, the residue Y having a reactive group may be directly added to the affinity peptide tag without providing the linker Y. In this case, by adding the amino acid derivative to the C or N terminus of the affinity peptide tag, it can be easily combined with the compound that forms the residue Z.

  In the present invention, a label molecule may be added to the linker Y. By introducing the label molecule, the target substance can be easily detected with high sensitivity. As the labeling molecule, for example, a fluorescent substance, a stable isotope, and a radioisotope can be used. The fluorescent substance used in the present invention is not particularly limited, but examples include xanthene dyes such as fluorescein, rhodamine, or rhodol, cyanine dyes, coumarin dyes, porphyrin dyes, and complex dyes thereof. Examples of the labeling molecule include isotope elements, deuterium, tritium, 15N nitrogen isotope, and 13C or 14C carbon isotope. For example, a labeled molecule can be easily introduced into the linker Y by adding an amino acid containing an isotope element such as deuterium. If necessary, the affinity peptide tag portion may be labeled with these label molecules.

  Further, by using an amino acid or a peptide as the linker Y, a protease recognition site for separating the target substance and the affinity tag can be easily introduced. By the protease that recognizes the recognition site, the functional tag bound to the target substance can be divided into the affinity peptide tag and the target substance, and the target substance can be concentrated. The protease used in the present invention is not particularly limited as long as it selectively cleaves the peptide bond of the amino acid between linker Y or linker Y and reactive group Z.

Examples of proteases used in the present invention include chymotrypsin A (C-terminal side of aromatic amino acid residue), endoproteinase Glu-C (protease V8), endoproteinase
Lys-C, Factor Xa (C-side of Arg residue), Plasmin (C-side of Arg, Lys residue), Thrombin (C-side of Arg residue) and Trypsin (C-terminus of Arg, Lys residue) Serine proteases such as serine protease, sermolysin (hydrophobic L-amino acids (especially C-terminal side of Ile, Leu, Val, Phe, Met, Ala) residues), disperse (Leu-Phe bond), endoproteinase
Arg-C, aminopeptidase M (N-terminal side of L-amino acid with free α-amino group or α-imino group), carboxypeptidase B (C-terminal Arg, Lys, Orn residues), and carboxypeptidase Y (hydrolyzes C-terminal L-amino acids of proteins and peptides. Pro decomposes).

  In addition, the reactive group of residue Z of the present invention refers to a structural portion that selectively binds to a target substance to be concentrated, detected, or purified, and includes not only ordinary chemical groups but also lipophilic residues. , Antibodies, haptens, antigens and ligands. When the reactive group is a lipophilic residue containing a long carbon chain, an antibody, or the like, the reactive group constitutes the residue Z as it is. Here, the ligand refers to a substance that specifically binds to a target substance such as a protein.

  Examples of the reactive group (chemical group) of the present invention include O, S and NH. In this case, the residue Z of the present invention can be represented by -A-B-A-, wherein each A is independently O, S or NH, and B is substituted or unsubstituted C1-20. Linear, branched or cyclic alkyl, aralkyl, aryl, alkaryl, alkenyl, alkynyl, alkoxy, alkenyloxy, alkynyloxy, heteroalkyl, heterocyclic, alkyl-heterocyclic or heterocyclic-alkyl be able to.

  When various proteins are used as target substances, a reactive group that binds to a functional group of the protein, for example, an amino group, an aldehyde group, or an SH group is selected. For example, an active ester group that binds to a free amino group (-NH2) such as an ε-amino group, a maleimide group that binds to a thiol group (-SH) such as cysteine, or an aldehyde group (-CHO) of an oxidatively damaged protein Such as hydrazide binding.

  In order to add the reactive group (chemical group), the linker Y can be bound to the C-terminus or N-terminus of the affinity peptide tag, and the residue Z having the reactive group is linked to the linker Y or direct affinity. To the sex peptide tag. Examples of compounds that can be used to form the residue Z include the following.

Next, the manufacturing method of the functional tag of this invention is demonstrated.
The affinity peptide tag of the present invention can be produced by incorporating a nucleic acid molecule encoding the peptide tag into an appropriate vector for expression, or by a chemical synthesis method. In the case of chemical synthesis, amino acids are sequentially bound to a carrier for synthesis, followed by linker Y and residue Z, and residues Z and linker Y are bound to an appropriate carrier, and amino acids are bound thereto. You may manufacture by any method of combining sequentially.

  For the production of the affinity peptide tag of the present invention, commercially available resins for peptide / protein synthesis can usually be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl. Examples include phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can.

  Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the peptide peptide of interest and according to various known condensation methods. When the functional tag of the present invention does not require the linker Y, a residue Z is introduced by reacting a predetermined compound with the last amino acid of the peptide sequence. Further, when the linker Y is composed of an amino acid or a derivative thereof, the linker Y is condensed on the resin following the peptide tag according to various condensation methods known per se. The compound is reacted to introduce residue Z. At the end of the reaction, the functional tag of the present invention is cut out from the resin, and at the same time, various protecting groups are removed. If necessary, an intramolecular disulfide bond forming reaction is performed in a highly diluted solution to obtain the desired functional tag. .

  With respect to the condensation of the above-mentioned protected amino acids, various activating reagents used for peptide / protein synthesis can be used, and carbodiimides are particularly preferred. Examples of the carbodiimide include DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (for example, HOBt, HOBt), or the protected amino acid is activated in advance as a symmetric anhydride, HOBt ester or HOOBt ester. It can later be added to the resin.

  The solvent used for the activation of the protected amino acid and the condensation with the resin is appropriately selected from solvents that can be used for the peptide / protein condensation reaction. For example, N, N-dimethylformamide, N, N-dimethylacetamide, acid amides such as N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl Sulfoxides such as sulfoxide, ethers such as pyridine, dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or a mixture thereof can be used.

  The reaction temperature is appropriately selected from the range suitable for the protein bond forming reaction, but is usually in the range of -20 ° C to 50 ° C. The activated amino acid derivative is usually used in an excess of 1.5 to 4 times. When a test using the ninhydrin reaction is performed and the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. If sufficient condensation is not obtained even after repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.

  Examples of the protective group for the amino group of the raw material include Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl. , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc, etc. are used.

  The protection of the carboxyl group of the amino acid is, for example, alkyl esterification (eg, linear, branched, such as methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl). Or cyclic alkyl esterification), aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl It is carried out by hydrazide, tertiary butoxycarbonyl hydrazide, trityl hydrazide and the like.

  Moreover, protection of the hydroxyl group of serine can be performed by, for example, esterification or etherification. Examples of groups suitable for esterification include groups derived from carbonic acid such as lower alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, benzyloxycarbonyl groups, and ethoxycarbonyl groups. Examples of groups suitable for etherification include benzyl, tetrahydropyranyl, and t-butyl groups.

  Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl2-Bzl, 2-nitrobenzyl, Br-Z, and tertiary butyl. Examples of the protecting group for imidazole of histidine include Tos, 4-Methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like can be mentioned.

  Examples of methods for removing (eliminating) protecting groups include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfone. Acid treatment with an acid, trifluoroacetic acid, or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like. The elimination reaction by the acid treatment is generally performed at a temperature of about −20 ° C. to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4 -Addition of a cation scavenger such as butanedithiol or 1,2-ethanedithiol is effective. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the 1,2-ethanedithiol or 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it can be removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like. Protection of a functional group that should not participate in the condensation reaction, protection group, elimination of the protective group, activation of a functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.

The functional tag of the present invention formed on the carrier is removed from the carrier by treatment with a known solvent and a releasing agent, and is obtained in a solid or powder state by a freeze drying method or the like. Can do.
A method for detecting a target substance using the functional tag of the present invention will be described. In the method of the present invention, the functional tag is mixed with a sample containing a target substance, and the mixture or suspension is contacted with a carrier that holds a metal ion that binds to the affinity peptide tag of the functional tag. And then recover the functional tag from the carrier.

When the target substance is a biomolecule such as protein or saccharide, blood, body fluid, or tissue extract collected from an animal such as a human is treated with an appropriate solution and used as a sample.
Moreover, the metal ion having affinity for the functional tag of the present invention is not particularly limited as long as it has affinity for the peptide tag. As examples of the metal ions, divalent metal ions such as Cu2 +, Zn2 +, Ni2 +, Ca2 +, Co2 +, Mg2 +, and particularly Ni2 + ions are preferable. Usually, the carrier carrying these metal ions is not particularly limited, but various gels, particularly agarose gels, are preferably used. The metal ion-carrier that captures the affinity peptide tag can be prepared by chelating with the desired metal ion coordinated on the gel. As the support for fixing the gel, a glass plate, a plastic plate, a 96-well plate, or the like can be used. In particular, by using a 96-well plate in which the chelated metal ion-gel is formed at the bottom of the well, the detection of the target substance can be easily performed.

  The binding force between the metal ion-immobilized carrier and the affinity peptide tag depends on the type of coordinated metal ion and the pH of the solution. In the present invention, the pH of the solution is usually 6 to 9, particularly preferably 7.5 to 8.5. On the other hand, since the binding force between the metal ion-immobilized carrier and the affinity peptide tag is hardly affected by the surfactant or the high salt concentration buffer, it is suitable for purification of membrane proteins that require them. In addition, the functional tag-target substance complex of the present invention captured on the carrier is recovered by adding a chelating agent, acid, or alkali to separate the bond between the affinity peptide tag and the metal ion. Can do. The target substance can be separated and recovered from the carrier-functional tag complex by allowing a predetermined protease to act on the protease cleavage site of linker Y.

  Collected target substances can be anion exchange HPLC (high performance liquid chromatography), liquid chromatography mass spectrometer, column chromatography, polyacrylamide gel electrophoresis, reverse phase chromatography, ion pair chromatography, affinity chromatography, etc. Can be used for identification and quantification.

  Furthermore, the functional tag of the present invention is mixed with a sample containing a protein, and the mixture is contacted with a carrier that retains metal ions that bind to the affinity peptide tag of the functional tag, and then the target from the carrier. By recovering the functional tag bound to the molecule, the target protein can be purified.

  Such a purification method not only can easily obtain biomolecules such as hormones and enzymes that exist in trace amounts in the living body, but also makes it possible to easily and efficiently extract rare proteins produced by genetic recombination. . The target protein obtained using the functional tag of the present invention is further subjected to secondary purification by column chromatography, polyacrylamide gel electrophoresis, reverse phase chromatography, ion pair chromatography, affinity chromatography, etc. Can do.

  Next, the present invention will be described in detail and specifically based on examples. The examples are intended to illustrate the present invention and are not intended to limit the protection scope of the present invention.

  A functional tag having an oxidative damage protein as a target substance was produced, and the method for detecting a target substance of the present invention was carried out using the functional tag. Non-physiological post-translational modification (oxidative damage) of proteins induced by reactive oxygen in the body, especially aldehyde formation of arginine, lysine, and proline residues is not limited to aging but is associated with many diseases It has been reported that In this example, a functional tag was prepared by adding a hydrazide covalently bonded to an aldehyde group to the C-terminal side of the affinity tag, and the aldehyded protein was purified and identified.

Example 1 Production of Functional Tag (I) Using Oxidative Damage Protein as Target Substance In this example, the following functional tag (I) was produced.
HGHAHGHAHGHAHGHGHAHG-EGAGA-hydrazide (I)
Affinity peptide tag HGHAHGHAHGHAHGHGHAHG (SEQ ID NO: 1)
Linker Y EGAGA (SEQ ID NO: 2)
Hydrazide -NH (CH (CH3)) CO (NH (NH2))
In this example, the functional tag (I) was manufactured as follows. First, polystyrene chlorotrityl chloride resin (BeadTech, 3.0 g, 1.3 mmol / g, 3.9
mmol) was shaken in a solution of anhydrous N-methyl-2-pyrylolidone (NMP) (15 mL) containing hydrazine (10 eq.) at room temperature for 10 hours. Filter this resin through NMP
The trihydrazine resin 1 was obtained by washing 3 times with 20 mL and drying under reduced pressure. The synthesis of the affinity peptide tag was performed manually using this trihydrazine resin 1.

The Fmoc amino acid used for the synthesis was used as a 0.5 M NMP solution. The coupling reagent was prepared in advance as an NMP (0.5 M) solution, and the activator N, N′-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) were prepared at a concentration of 2M. In all the steps, 10 equivalents of an amino acid and a coupling reagent were used with respect to trihydrazine resin 1, and one coupling reaction was performed for 2 hours. Fmoc deprotection was performed using 20% piperidine-NMP solution. In addition, TFA / H2O (95: 5) was used to cleave the peptide from the resin and deprotect the amino acid side chain.
In v / v), the resin was treated for 3 hours. The resin was washed with TFA and the filtrate was concentrated by vacuum distillation. Diethyl ether was added to this solution to precipitate the crude peptide, collected by centrifugation, dissolved in H 2 O and lyophilized. The yield of the peptide was determined by Shimadzu
High performance liquid chromatography (Shimadzu HPLC) using Shim-pack C-18 column (250 × 4.6 mm, 5μm, 100Å, HPLC flow rate 1 mL / min)
SCL-10A (UV (230 nm) detector)). Yield 7.4% (MALDI-TOF [M + H] + calcd 2415.8, found
2416.3).

Example 2 Production of Functional Tag (II) Using Oxidative Damage Protein as a Target Substance In this example, the following functional tag (II) was produced.
HGHAHGHGHAHGEGAGA-hydrazide (II)
Affinity peptide tag HGHAHGHGHAHG (SEQ ID NO: 3)
Linker Y EGAGA (SEQ ID NO: 2)
Hydrazide -NH (CH (CH3)) CO (NH (NH2))
The functional tag was produced and recovered in the same manner as in Example 1 except that the affinity peptide tag of SEQ ID NO: 1 was changed from 10 histidines to 10 and the number of histidines was 6 in SEQ ID NO: 3.

Example 3 Production of Functional Tag (III) Using Abnormally Expressed Protein as Target Substance In this example, the following functional tag (III) was produced.
Iodoacetamide-GAGAE-GHGHAHGHGHAH (III)
Affinity peptide tag GHGHAHGHGHAH (SEQ ID NO: 4)
Linker Y AGAGE (SEQ ID NO: 5)
Iodoacetamide -NHCOCH2I
In this example, based on the following scheme, the functional tag (III) was produced and recovered by the following method.

In this example, all amino acids used had an L-type structure unless otherwise specified. Fmoc-protected amino acids were purchased from Novabiochem (Darmstadt, Germany). Glu the side chain protection of Fmoc-protected amino acids
(OtBu) and His (Boc). All other reagents and solvents are grades used for analysis and peptide synthesis, and these are from Merck (Darmstadt, Germany), B
& J Bioscience and Aldrich. All solvents and reagents used in the synthesis and analysis were analytical grade. All HPLC analyzes are RP18 (Shim Pack)
This was performed using a Shimadzu SCL-10A VP equipped with a 250 mm × 5 mm column and a UV detector (230 nm). Characterization of the product was performed using MALDI-TOF (KRATOS analysis Axima-CFR, Shimadzu group).

The peptide of this example was prepared by the stepwise fluoren 9-ylmethoxycarbonyl (Fmoc) solid phase method as shown in the following scheme. First, Fmoc amino acid, 0.5
Stored as M NMP solution, coupling reagent was pre-dissolved in NMP (0.5 M solution), while the activator DCC and HOBt were made into 2M solutions. In all cases, 10 equivalents of amino acid and coupling reagent were used, and a 2 hour coupling was performed once. Fmoc deprotection was performed using 20% piperidine in NMP. N-terminal iodoacetic acid coupling was performed by conventional DCC coupling. Peptide cleavage from the resin of amino acid side chains by TFA / H2O
(95: 5 v / v) for 3 hours. The resin was washed with TFA and the filtrate was partially evaporated. The crude product was precipitated with diethyl ether, collected by centrifugation, dissolved in H2O and lyophilized. The yield of the peptide was measured using a Shimadzu Simpack C-18 column (250 mm
Using an SCL-10A VP Shimadzu apparatus equipped with a UV detector (230 nm), an X 4.6 mm column, 5 μm, 100 mm, flow rate 1 ml / min) was used. The result is 15% yield and MALDI-TOF
[M + H] + found 1717.8.

(Example 4) Separation and Purification of Oxidized Peptide An angiotensin (hereinafter abbreviated as AngII), which is a peptide having the amino acid sequence of SEQ ID No. And attempted to purify it.

AngII: DRVYIHPF (SEQ ID NO: 6)
First, an aqueous solution (1) having an AngII concentration of 200 μM was prepared. Next, 10 μl of an aqueous solution having a NaOCl concentration of 120 μM was added to 10 μl of the AngII solution, and left at 37 ° C. for 30 minutes to prepare a solution (2) of oxidized AngII (hereinafter abbreviated as OxAngII). Next, OxAngII solution (2)
To the powder obtained by freeze-drying 20 μl, add 10 μl of 6M guanidine hydrochloride solution containing 4 mM of functional tag (II) obtained in Example 2 and react at 67 ° C. for 10 minutes. An OxAngII solution (3) to which (II) was added was prepared. Thereafter, the unreacted functional tag (II) in the OxAngII solution (3) was removed using a chip-type micro reversed phase column (Zip-TipC18, Millipore). The affinity tag (II) hardly adsorbs on the reverse phase column in H2O containing 0.05% formic acid. Taking advantage of this property, pass OxAngII solution (3) through Zip-TipC18, and then wash Zip-TipC18 with H2O containing 0.05% formic acid, so that only OxAngII with the functional tag (II) added is retained and unreacted. The functional tag (II) was successfully removed. Then, OxAngII with functional tag (II) retained in the column was added to 70% acetonitrile (containing 0.05% formic acid)
Elute with 20 μl.

1x Tris buffer (pH 9) is added to OxAng II with functional tag (II) eluted from the column to adjust the pH of solution (3) to 7.5 or higher, and this solution (3) carries chelated Ni2 + ions. Magnetic beads (trade name Ni-NTA
Magnetic Agarose Beads, QIAGEN USA) for 30 minutes at 4 ° C, washed 3 times with Tris buffer (pH 8.0), and then washed with functional tag (II) from magnetic beads using H2O containing 0.1% TFA. The added OxAngII was eluted. A solution (4) containing OxAngII thus purified and concentrated was prepared. Subsequently, AngII in each state was subjected to liquid chromatography mass spectrometry (LC-MS, mass spectrometry:
LCQDECA (Thermo Electron, Germany), HPLC: Nanospace SI-2 (Shiseido))).

  The measurement results will be described with reference to FIGS. 1 and 2 show the results of measuring the Ang II solutions (1) to (4) in each state with a liquid chromatography mass spectrometer (LC-MS). Fig. 1 is an MS chromatogram, and Figs. 2 (1) to (4) show the peptides detected at the residence times of 19.5 to 22.5 minutes (dotted square area) in Fig. 1 (1) to (4), respectively. MS spectrum.

Since (1) in FIG. 1 is a single state containing untreated AngII, one sharp chromatogram is detected. AngII has a molecular weight of about 1045.5. Reflecting this, in the MS spectrum (1) of FIG. 2, the molecular weight / ion value (m / z) is about 1046.5.
A spectrum that is monovalently ionized at (a1 +) and a spectrum that is divalently ionized at m / z of about 524.7 (a2 +) are observed.

The AngII solution (2) contains artificially oxidized AngII (OxAngII). As this state changes, the MS chromatogram (2) in Fig. 1 shifts toward a slower residence time and splits into several. doing. The MS spectrum (2) in Fig. 2 is also m / z.
Reflects various oxidation states from 980 to 1140. The ▼ b1 + portion of the MS spectrum (2) corresponds to a monovalent ion, and the ▼ b2 + portion corresponds to a divalent ion.

  The AngII solution (3) is obtained by adding the functional tag (II) of the present invention prepared in Example 2 to the solution (2) and causing the reaction, and the retention in the chromatogram of FIG. 1 (3). A new peak is shown between about 20-21 minutes of time. In addition, divalent to hexavalent ions are observed in the portions indicated by ▼ c2 + to ▼ c6 + in the MS spectrum of FIG. By adding the functional tag (II) of the present invention (molecular weight about 1640) to OxAngII having a molecular weight of about 980 to 1140, the molecular weight becomes about 2620 to 2780, and the divalent ion of the molecule has an m / z of about 1310 to 1390. , Trivalent ions are about 870-930. Since not all of OxAngII observed in FIG. 1 (2) is aldehyded, the number of spectra of the functional tag (II) adduct newly observed in FIG. 2 (3) decreases. However, the approximate value and the m / z of the observed spectrum were in agreement.

  The AngII solution (4) is obtained by purifying OxAngII, to which the functional tag (II) is added, from the sample of the AngII solution (3) using a Ni column. The spectrum derived from OxAngII (▼ b1 + and ▼ b2 +) observed in Fig. 2 (3) disappeared, and the spectrum derived from functional tag (I) -OxAngII newly observed in Fig. 2 (3) (▼ c2 + ~ ▼ c6 +) only. Therefore, it can be seen that by using the functional tag (II) of the present invention, aldehyde-ized peptides in various oxidation states that bind to the functional tag (II) were purified.

Example 5 Addition of Functional Tag (I) to Oxidized State Peptide and Cleavage Removal of Affinity Peptide Tag Part Similarly to Example 4, AngII was artificially damaged by oxidation, and aldehyde-modified OxAngII was The functional tag (I) was added to the OxAngII, and the affinity peptide tag portion was cleaved off.

AngII: DRVYIHPF (SEQ ID NO: 6)
Functional tag (I): HGHAHGHAHGHAHGHGHAHG-EGAGA-hydrazide First, an aqueous solution (1) having an AngII concentration of 200 μM was prepared. Next, 10 μl of an aqueous solution having a NaOCl concentration of 120 μM was added to 10 μl of the AngII solution, and the mixture was allowed to stand at 37 ° C. for 30 minutes to prepare an OxAngII solution (2). Next, OxAngII solution (2)
To the powder obtained by freeze-drying 20 μl, add 10 μl of 6M guanidine hydrochloride solution containing 4 mM of functional tag (II) obtained in Example 1 and react at 67 ° C. for 10 minutes. An OxAngII solution (3) to which (II) was added was prepared. Thereafter, the unreacted functional tag (II) in the OxAngII solution (3) was removed using a chip-type micro reversed phase column (Zip-TipC18, Millipore). The affinity tag (II) hardly adsorbs on the reverse phase column in H2O containing 0.05% formic acid. Taking advantage of this property, pass OxAngII solution (3) through Zip-TipC18, and then wash Zip-TipC18 with H2O containing 0.05% formic acid, so that only OxAngII with the functional tag (II) added is retained and unreacted. The functional tag (II) was removed. Thereafter, OxAngII to which the functional tag (II) retained on the column was added was eluted with 20 μl of 70% acetonitrile (containing 0.05% formic acid).

Magnetic beads carrying the chelated Ni2 + ion (trade name Ni-NTA) with 1M Tris buffer (pH 9) added to OxAngII with functional tag (II) eluted from the column to a pH of 7.5 or higher
Magnetic Agarose Beads, QIAGEN, USA) was contacted at 4 ° C. for 30 minutes and washed 3 times with Tris buffer (pH 8.0).

Add 20 μl of 100 mM ammonium acetate solution to this magnetic bead, and then add 10 μl of V8 protease (endoproteinase Glu-C) solution, and perform the cleavage reaction at 25 ° C. for about 18 hours to remove the functional tag (I) and purify. Then, a solution (2) containing concentrated OxAngII was prepared. Subsequently, AngII in the solutions (1) and (2) was subjected to liquid chromatography mass spectrometry (LC-MS,
Mass spectrometry: Measured with LCQDECA (Thermo Electron, Germany), HPLC: Nanospace SI-2 (Shiseido)). The cleavage site of V8 protease is the glutamic acid residue (E) at the left end of the linker (EGAGA) of the functional tag (I) and the affinity peptide tag.

The measurement results will be described with reference to FIGS. FIG. 3 shows a liquid chromatography mass spectrometer (LC-MS, mass spectrometry: LCQDECA (manufactured by Thermo Electron,
Germany), HPLC: MS chromatogram measured by Nanospace SI-2 (manufactured by Shiseido).
FIG. 3 shows the MS chromatogram intensity (1) of functional tag (I) -added OxAngII (before cleavage) and the OxAngII MS chromatogram (2) after cleavage of functional tag (I) at each residence time. Also, (1) and (2) in FIG. 4 are MS spectra of the peptides measured at residence times 9.5 to 12 minutes (portions surrounded by broken lines) in FIGS. 3 (1) and (2), respectively.

The molecular weight of the functional tag (I) is about 2415.8, and when it is digested with V8 protease, it is hydrolyzed into an affinity peptide tag having a molecular weight of about 2145.65 and a linker Y moiety having a molecular weight of about 288.15. In addition, OxAngII produced in this example is AngII accompanied by aldehyde formation of an arginine residue and has a molecular weight of about 1000.6. When a functional tag (I) is added to the OxAngII, the molecular weight is about 3398.4.
A functional tag (I) -OxAngII of (2415.8 + 1000.6-18) is generated. Here, -18 in the calculation formula is the molecular weight of water molecules that are generated and separated when the aldehyde and the hydrazide of the functional tag (I) are combined.

  Furthermore, when functional tag (I) -OxAngII is digested with V8 protease, it becomes OxAngII having a molecular weight of about 1270.8 (3398.4-2145.7 + 18). +18 in the above formula is the molecular weight of a water molecule added when the glutamic acid residue (E) of the functional tag (I) is hydrolyzed at the C-terminus by V8 protease. Table 1 summarizes the molecular weight of the product after each reaction and the m / z of the observed MS spectrum. In addition, ▼ a2 + to ▼ a8 + in FIG. 4 (1) show the spectrum (bivalent to octavalent) of affinity peptide tag-added-OxAngII, and symbols ▼ b1 + to ▼ b3 + in FIG. The OxAngII spectrum (monovalent to trivalent) is shown after cleaving affinity peptide tagging-OxAngII. In both FIGS. 4 (1) and 4 (2), the molecular weights agree with the calculated values (Table 1). Therefore, it can be seen that the affinity peptide tag of almost all functional tags (I) was removed by the affinity peptide tag cleavage treatment with V8 protease.

(Example 6) Detection of oxidative damage protein by stable isotope-labeled functional tag (I) When an unknown sample is analyzed by a mass spectrometer, it is important to discriminate a target protein. In this example, in order to distinguish an oxidatively damaged protein as a target protein, the protein was detected using a functional tag (I) labeled with a stable isotope-labeled amino acid.

  First, a functional tag (I) was produced in the same procedure as in Example 1, except that a linker Y moiety (GAGA) was formed using an amino acid labeled with a deuterium nucleus. The labeled functional tag (I) is referred to as functional tag (I-d10). In addition, the functional tag (I) that is not labeled is mixed with an equivalent amount of the functional tag (I-d0) and the functional tag (I-d10) and the functional tag (I-d0). It is written as sex tag (I-mix). Next, a solution with an Ang II concentration of 200 μM was prepared, 10 μl of an aqueous solution with a NaOCl concentration of 120 μM was added to 10 μl of the Ang II solution, and the solution was allowed to stand at 37 ° C. for 30 minutes to prepare an OxAng II solution. Next, 10 μl of 6M guanidine hydrochloride solution containing 4 mM of functional tag (I-mix) is added to the powder obtained by freeze-drying 20 μl of this solution, and reacted at 67 ° C. for 10 minutes. I-mix) -OxAngII was formed. The solution containing the functional tag (I-mix) -OxAngII was analyzed with a liquid chromatography mass spectrometer (LC-MS) to obtain an MS chromatogram and an MS spectrum.

  FIG. 5 shows an MS spectrum in which the functional tag (I-mix) -OxAngII was detected. Insets 5A, 5B, and 5C are enlarged views of the pentavalent (▼ 5 +), tetravalent (▼ 4 +), and trivalent (▼ 3 +) MS spectra, respectively. In this example, in the sample solution, a functional tag (I-d0) having a molecular weight of about 2415.8 and a functional tag (I-d0) having a molecular weight of about 2425.8 are added to OxAngII having a molecular weight of about 1000.6 by a dehydration reaction. In addition, a functional tag (I-d0) -OxAngII having a molecular weight of about 3398.4 and a functional tag (I-d10) -OxAngII having a molecular weight of about 3408.4 are generated. Table 2 shows the m / z of monovalent to octavalent ions observed in the MS spectrum. From the inset of FIG. 5, doublet spectra having molecular weight differences of about 3.3, 2.5, and 2.0 were observed for trivalent, tetravalent, and pentavalent ions, respectively.

  Therefore, it is clearly shown that the target protein in this example is an oxidative damage protein. In the present invention, by using a stable isotope-labeled amino acid in this manner, a labeled functional tag can be easily designed, and complicated post-translational modifications can be analyzed.

Example 7 Analysis of Abnormally Expressed Protein Using Functional Tag (III) Functional tag produced in Example 3 for an abnormally expressed protein having the amino acid sequence of SEQ ID NO: 7 (hereinafter abbreviated as pep7) The quantitative analysis was performed using (III).
Abnormally expressed protein: ATVRTFSC (SEQ ID NO: 7)
First, a pep7 solution (1) having a protein concentration of 10 μM was prepared. Next, 2 μl of 100 mM DTT was added to 98 μl of the solution (1) and incubated at 57 ° C. for 1 hour. Thereafter, 10 μl of a 4 mM solution of the functional tag (III) obtained in Example 3 was added and reacted at room temperature for 1 hour to add the functional tag (III) to prepare a pep7 solution (2). Subsequently, a liquid chromatography mass spectrometer (LC-MS,
Mass spectrometry: Measured with LCQDECA (Thermo Electron, Germany), HPLC: Nanospace SI-2 (Shiseido)).

The measurement results will be described with reference to FIGS. 6 and 7 show the results obtained by measuring the pep7 solutions (1) and (2) in each state with a liquid chromatography mass spectrometer (LC-MS). FIG. 6 is an MS chromatogram, and FIGS. 7 (1) and (2) show the residence times of 8.2 to 10 for FIGS. 6 (1) and 6 (2).
It is a MS spectrum of the peptide detected by minute (dotted line square area).

  Since (1) in FIG. 6 is one state including unprocessed pep7, one sharp chromatogram (▼ S1) is detected. Reflecting this, the molecular weight of pep7 is about 883.5, and in the MS spectrum (1) of FIG. 7, the spectrum that is ionized monovalently to molecular weight / ion valence (m / z) about 884.5 (▼ a1 +) A divalent ionized spectrum at m / z of about 442.8 (▼ a2 +) is observed.

  (2) in FIG. 6 is an MS chromatogram of the pep7 solution (2) to which the functional tag (III) is added. A new main peak appears at a residence time of about 8.5 minutes (▼ S2). This corresponds to pep7 to which the functional tag (III) is added. Divalent to hexavalent ions are observed in the portions indicated by ▼ b2 + to ▼ b6 + in the MS spectrum of FIG. Since the molecular weight of functional tag (III) is 1764.8, when pep7 reacts with this, iodine (atomic weight about 127) of functional tag (III) and hydrogen of SH group of cysteine residue of pep7 will be separated. The molecular weight of pep7 to which the functional tag (III) is added is 1764.8 + 883.5-128 = 2520.3. Therefore, m / z of divalent to hexavalent ions is about 1261.2, 841.1, 631.0, 505.1, and 421.1, respectively, which is consistent with the observed value. In FIG. 6 (2), there is also a peak derived from the product (▼ S3) generated by the mixture of pep7 (▼ S1), which has not reacted with the functional tag (III), and DTT and pep7. The respective peaks ▼ a1 +, ▼ a2 +, ▼ c1 +, and ▼ c2 + are also observed in the MS spectrum of 7 (2).

(Explanation of sequence listing)
The sequence numbers in the sequence listing in the present specification indicate the following sequences.
(SEQ ID NO: 1) This is the amino acid sequence of the affinity peptide tag used in the production of the functional tag (I) with the oxidative damage protein as a target substance in Example 1.
(SEQ ID NO: 2) This is the amino acid sequence of linker Y used in the production of functional tags (I) and (II) using oxidative damage proteins as target substances in Examples 1 and 2.
(SEQ ID NO: 3) This is the amino acid sequence of the affinity peptide tag used in the production of the functional tag (II) with the oxidative damage protein as a target substance in Example 2.
(SEQ ID NO: 4) This is the amino acid sequence of the affinity peptide tag used in the production of the functional tag (III) with the oxidative damage protein as a target substance in Example 3.
(SEQ ID NO: 5) This is the amino acid sequence of linker Y used in the production of functional tag (III) using oxidative damage protein as a target substance in Example 3.
(SEQ ID NO: 6) This is the amino acid sequence of angiotensin (AngII) used in Examples 4 and 5 to create an oxidative damage peptide.
(SEQ ID NO: 7) This is the amino acid sequence of the abnormally expressed protein analyzed in Example 7 using the functional tag (III).

FIG. 1 is an MS chromatogram obtained by measuring AngII solutions (1) to (4) in each state in Example 4 using a liquid chromatography mass spectrometer. FIG. 2 shows the MS spectra of the peptides detected at the residence times of 19.5 to 22.5 minutes (dotted square region) in FIGS. 1 (1) to (4), respectively. FIG. 3 shows the MS chromatogram intensity (1) of functional tag (I) -added OxAngII (before cleavage) and functional tag (I) after cleavage measured in a liquid chromatography mass spectrometer in Example 5. The MS chromatogram (2) of OxAngII is shown. FIG. 4 shows MS spectra (1) and (2) of the peptides detected at residence times of 9.5 to 12 minutes (parts surrounded by broken lines) in FIGS. 3 (1) and (2), respectively. It is. FIG. 5 shows the MS spectrum in which the functional tag (I-mix) -OxAngII was detected in Example 6. Insets 5A, 5B, and 5C are enlarged views of the pentavalent (▼ 5 +), tetravalent (▼ 4 +), and trivalent (▼ 3 +) MS spectra, respectively. FIG. 6 is an MS chromatogram obtained by measuring the pep7 solutions (1) and (2) in each state with a liquid chromatography mass spectrometer (LC-MS). FIG. 7 is an MS spectrum of the peptide detected at a residence time of 8.2 to 10 minutes (dotted square region) in FIGS. 6 (1) and (2), respectively.

Claims (22)

  A functional tag comprising an affinity peptide tag, a linker Y, and a residue Z having a reactive group, wherein the affinity peptide tag has at least three (X-His) units, and X is any amino acid Or the functional tag, which is an amino acid derivative.   The functional tag according to claim 1, wherein the affinity peptide tag has at least 4 (X-His) units.   A functional tag consisting of an affinity peptide tag and a residue Z having a reactive group, wherein the affinity peptide tag has at least three (X-His) units, and X is any amino acid, or The functional tag, which is an amino acid derivative.   4. A functional tag according to claim 3, wherein the affinity peptide tag has at least 4 (X-His) units. A functional tag comprising an affinity peptide tag and a reactive group Z and represented by the following formula (1):
(His) l- (X-His) m- (X) n- (Y) pZ (1)
(Wherein, X is any amino acid or amino acid derivative except histidine, Y is a linker, l is 0 or 1, m is an integer of 4 to 100, n is 0 or 1, and p is 0. Or 1).   X is an amino acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan and tyrosine. The functional tag according to claim 5.   The functional tag according to claim 5, wherein X is an amino acid selected from the group consisting of cysteine and tryptophan.   The functional tag of Claim 5 whose m is an integer of 5-25 in Formula (1).   The functional tag of Claim 5 whose m is an integer of 6-15 in Formula (1).   The functional tag according to claim 5, wherein the linker Y is a peptide sequence containing 3 to 30 amino acids and / or amino acid derivatives.   The functional tag according to claim 1, wherein the linker Y includes a label molecule.   The functional tag according to claim 1, wherein the reactive group of residue Z is histidine. The functional tag according to claim 5 represented by the following formula (2):
HGHAHGHAHGHAHGHGHAHG-EGAGA-NH (CH (CH3)) CO (NH (NH2)) Formula (2). The functional tag according to claim 5 represented by the following formula (3):
HGHAHGHGHAHG-EGAGA-NH (CH (CH3)) CO (NH (NH2)) Formula (3). The functional tag according to claim 5 represented by the following formula (4):
ICH2CONH-GAGAE-GHGHAHGHGHAH formula (4).   6. The function according to claim 1, wherein the affinity tag and / or the linker Y is labeled with a fluorescent substance, a stable isotope element, or a radioisotope. Sex tag.   A protein detection kit comprising the functional tag according to claim 1.   A protein purification kit comprising the functional tag according to claim 1.   A carrier that holds a metal atom that mixes the functional tag according to any one of claims 1, 3, and 5 with a sample containing a target substance and binds the mixture to an affinity peptide tag of the functional tag. And then recovering the functional tag from the carrier, and a method for detecting a target substance.   20. The method according to claim 19, further comprising the step of cleaving the linker of the functional tag with a protease.   The functional tag according to any one of claims 1, 3, and 5 is mixed with a sample containing a protein, and the mixture is supported with a carrier that holds a metal atom that binds to the affinity peptide tag of the functional tag. A method for purifying a protein, comprising contacting and then recovering the functional tag from the carrier.   The method according to claim 21, further comprising the step of cleaving the linker of the functional tag with a protease.