JPWO2013084526A1 - Biotin compound, biotin labeling agent, protein assembly - Google Patents

Abstract

The subject of this invention is producing the protein assembly which has a function. And a biotin compound effective for producing the same, and a solution means thereof is the following formula (1) [Formula 1] (wherein n is 1 or 2, and X is O Or X when n is 2 may be the same or different, R1 is a direct bond or an amino acid residue, and the amino acid of R1 when n is 2 The residues may be the same or different, and R2 is a direct bond when n is 1, and the amino acid residue of R2 when n is 2 is from the amino group at the end of the side chain. It is an amino acid residue having an amino residue from which one hydrogen atom has been removed, and the amino residue and the carboxylic acid residue of R1 are peptide-bonded, and R3 is a direct bond or an amino acid residue Each of the amino acid residues of R1, R3 is one amino acid residue, Is to provide a biotin compound represented by two or more amino acid residue is an amino acid residue is a peptide bond.).

Description

  The present invention relates to a biotinylated compound, a biotin labeling agent, and a protein assembly.

  Protein self-assembly plays an important role in nature, as exemplified by the formation of viruses, microtubules, flagella, actin fibers, and the like (Non-patent Document 1). The elucidation and establishment of such a self-assembling system gives insights into the design of biomaterials with unprecedented functions, and has attracted particular attention both in the biotechnology field and in the nanotechnology field. (Non-Patent Document 2).

  Although the design of new self-assembled structures using proteins as building blocks is still a challenging field, many recent studies have revealed some of them (non-patented) Reference 3). In the field of protein self-assembly, it is the construction of an assembly that not only makes it an assembly but also has a function as such as an enzyme or an antibody and can express that function (non-patented). Reference 4). Examples of the significance of the functional assembly include an increase in local concentration, an increase in the amount of immobilization during protein immobilization, and an increase in detection sensitivity when used in a detection system. For a combination of proteins having different functions, for example, in a combination of different enzymes, if it can be applied to a conjugation reaction in which the product of one enzyme becomes a substrate for the other enzyme, a highly efficient complex enzyme system can be constructed. . Therefore, if the functional self-assembly can be rationally designed, its usefulness becomes immeasurable.

  A transglutaminase derived from a microorganism that is a cross-linking enzyme (hereinafter referred to as MTG in the present specification) is used in a site-specific ligand labeling technique. MTG is a kind of cross-linking enzyme that catalyzes the acyl transfer reaction between glutamine (Q) and lysine (K) or primary amine, and is an extremely useful tool for post-translational modification of proteins. Since such a crosslinking reaction using MTG proceeds specifically with respect to the MTG recognition sequence in the protein, it can be easily modified without impairing the original function of the protein.

  Alkaline phosphatase (hereinafter sometimes referred to as AP in the present specification) is a dephosphorylation enzyme that hydrolyzes a phosphate ester under alkaline conditions, and dephosphorylates a terminal base of DNA; ELISA, Western blotting, etc. It is widely used as a labeling enzyme for antibody immune reactions.

C. Branden and J.M. Toe, Introduction to Protein Structure, 2nd ed. , Garland Publishing, New York, 1999. T.A. O. Yeasts and J.M. E. Padilla, Curr. Opin. Chem. Biol. , 2002, 12, 464. C. M.M. Niemyer, M.M. Adler, S.M. Gao and L.M. Chi, Angew. Chem. Int. Ed. , 2000, 39, 3056. T.A. F. Chou, C.I. So, B.B. R. White, J. et al. C. T.A. Carlson, M.M. Sarikaya and C.I. R. Wagner, ASC Nano, 2008, 2, 2519.

The first problem in self-assembly of proteins is selection of driving force for assembly. For example, non-covalent bonds such as π-π stacking, hydrogen bonding, and van der Waals force are used for self-assembly. However, due to its versatility and applicability, certain receptors and their ligands can be used. Emphasis is placed on the self-assembly design used. In particular, problems have arisen in the process of labeling ligands to proteins. This is because it is generally difficult to introduce a ligand into a target site of a protein.
The simplest labeling method is the chemical modification method. However, since the modification is aimed at the reactive sites on the protein surface, the function is unavoidable and the reaction is difficult to control and the place to be labeled is random. Therefore, it is not suitable for use in controlled self-assembly.

  From the above, it is indispensable for the construction of a controllable self-assembly system to modify the ligand without impairing the original three-dimensional structure of the protein.

  In addition, development of a modifying agent suitable for such modification is required.

  In order to solve the above-mentioned problems, the present inventor has made extensive studies and found that a specific biotin compound is biotinylated with a transglutaminase that recognizes a specific amino acid sequence. . In addition, it has been found that a biotinylated protein or peptide can be efficiently assembled into an aggregate, and that such an aggregate exhibits a greatly improved function compared to the function exhibited by the protein or peptide. .

  The present invention has been completed on the basis of such knowledge, and widely encompasses the inventions of the embodiments described below.

（式中、
ｎは、１又は２であり、
Ｘは、Ｏ又はＮＨであり、前記ｎが２である場合のＸは、同一であっても異なっていてもよく、
Ｒ^１は、直接結合またはアミノ酸残基であり、前記ｎが２である場合のＲ^１のアミノ酸残基は、同一であっても異なっていてもよく
Ｒ^２は、ｎが１の場合、直接結合であり、前記ｎが２の場合のＲ^２のアミノ酸残基は、側鎖の末端のアミノ基から１つの水素原子が除かれたアミノ残基を有し、且つ該アミノ残基とＲ^１のカルボン酸残基がペプチド結合しているアミノ酸残基であり、
Ｒ^３は、直接結合又はアミノ酸残基であり、
Ｒ^１又はＲ^３のアミノ酸残基は、それぞれ１個のアミノ酸残基であるか、又は２個以上のアミノ酸残基がペプチド結合しているアミノ酸残基である。）
で表わされるビオチン化合物。Item 1
Following formula (1)

(Where
n is 1 or 2,
X is O or NH, and when n is 2, X may be the same or different;
R ¹ is a direct bond or an amino acid residue, and the amino acid residue of R ¹ when n is 2 may be the same or different. R ² is a direct bond when n is 1. The amino acid residue of R ² in the case where n is 2 has an amino residue obtained by removing one hydrogen atom from the terminal amino group of the side chain, and the amino residue and R ¹ Is an amino acid residue having a peptide bond,
R ³ is a direct bond or an amino acid residue;
Each amino acid residue of R ¹ or R ³ is one amino acid residue, or an amino acid residue in which two or more amino acid residues are peptide-bonded. )
A biotin compound represented by

Item 2
Item 2. The biotin compound according to Item 1, wherein the amino acid residue of R ¹ or R ³ comprises at least one selected from the group consisting of a glycine residue and a leucine residue.

Item 3
Item 3. The biotin compound according to Item 1 or 2, wherein the amino acid residue of R ² is a lysine residue.

Item 4
The R ¹ amino acid residue is an amino acid residue in which 6 or less amino acid residues are peptide-bonded, or the R ³ amino acid residue is an amino acid residue in which 6 or less amino acid residues are peptide-bonded Item 4. The biotin compound according to any one of Items 1 to 3, which is a group.

（式中、
ｎは、１又は２であり、
Ｘは、Ｏ又はＮＨであり、前記ｎが２である場合のＸは、同一であっても異なっていてもよく、
Ｒ^１は、直接結合またはアミノ酸残基であり、前記ｎが２である場合のＲ^１のアミノ酸残基は、同一であっても異なっていてもよく
Ｒ^２は、前記ｎが１の場合、直接結合であり、前記ｎが２の場合のＲ^２のアミノ酸残基は、側鎖の末端のアミノ基から１つの水素原子が除かれたアミノ残基を有し、且つ該アミノ残基とＲ^１のカルボン酸残基がペプチド結合しているアミノ酸残基であり、
Ｒ^４は、直接結合又はアミノ酸残基であり、
Ｒ^１又はＲ^４のアミノ酸残基は、それぞれ１個のアミノ酸残基であるか、又は２個以上のアミノ酸残基がペプチド結合しているアミノ酸残基である。）で表わされる項１〜４の何れか１項に記載のビオチン化合物。Item 5
Following formula (2)

(Where
n is 1 or 2,
X is O or NH, and when n is 2, X may be the same or different;
R ¹ is a direct bond or an amino acid residue, and when n is 2, the amino acid residue of R ¹ may be the same or different. R ² is when n is 1, In the case where n is 2 and the amino acid residue of R ² is a direct bond, the amino acid residue of R ² has an amino residue obtained by removing one hydrogen atom from the terminal amino group of the side chain, and the amino residue and R ¹ carboxylic acid residue is an amino acid residue having a peptide bond,
R ⁴ is a direct bond or an amino acid residue;
Each amino acid residue of R ¹ or R ⁴ is one amino acid residue, or an amino acid residue in which two or more amino acid residues are peptide-bonded. The biotin compound according to any one of Items 1 to 4, which is represented by:

Item 6
Item 6. The biotin compound according to Item 5, wherein the amino acid residue of R ¹ or R ⁴ includes at least one glycine residue.

Item 7
Item 7. The biotin compound according to Item 5 or 6, wherein the amino acid residue of R ² is a lysine residue.

Item 8
The amino acid residue of R ^{1 is} an amino acid residue in which 6 or less amino acid residues are peptide-bonded, or the amino acid residue of R ^{4 is} an amino acid residue in which 5 or less amino acid residues are peptide-bonded Item 8. The biotin compound according to any one of Items 5 to 7, which is a group.

（式中、
Ｘは同一又は異なってＯ又はＮＨであり、
Ｒ^５ _、Ｒ^６、及びＲ^７は、それぞれ、同一又は異なって直接結合又はアミノ酸残基であり、
Ｒ^５ _、Ｒ^６、及びＲ^７のアミノ酸残基は、それぞれ１個、又は２個以上のアミノ酸残基がペプチド結合したアミノ酸残基である。）
で表わされる項１〜８の何れか１項に記載のビオチン化合物。Item 9
Following formula (3)

(Where
X is the same or different and is O or NH;
R ⁵ _, R ⁶ , and R ⁷ are the same or different and each is a direct bond or an amino acid residue;
The amino acid residues of R ⁵ _, R ⁶ , and R ⁷ are each an amino acid residue in which one or two or more amino acid residues are peptide-bonded. )
Item 9. The biotin compound according to any one of Items 1 to 8 represented by:

Item 10
Item 10. The biotin compound according to Item 9, wherein the amino acid residue of R ⁵ , R ⁶ , or R ⁷ includes at least one glycine residue.

Item 11
The R ⁵ amino acid residue is an amino acid residue in which 6 or less amino acid residues are peptide-bonded, and the R ⁶ amino acid residue is an amino acid residue in which 6 or less amino acid residues are peptide-bonded Item 11. The biotin compound according to Item 9 or 10, wherein the amino acid residue of R ^{7 is} an amino acid residue in which 5 or less amino acid residues are peptide-bonded.

で表わされる、項１に記載のビオチン化合物。Item 12
Following formula (4)

Item 2. The biotin compound according to Item 1, represented by:

で表わされる項５に記載のビオチン化合物。Item 13
Following formula (5)

Item 6. The biotin compound according to Item 5, represented by:

で表わされる項９に記載のビオチン化合物。Item 14
Following formula (6)

Item 10. The biotin compound according to Item 9, represented by:

Item 15
Item 15. A biotin labeling agent for a protein or peptide comprising the biotin compound according to any one of Items 1 to 14.

Item 16
Item 16. The biotin labeling agent according to Item 15, wherein the protein or peptide comprises one or more amino acid sequences represented by any of SEQ ID NOs: 1 to 4.

Item 17
Item 17. A protein or peptide biotin labeling kit comprising the biotin labeling agent according to Item 15 or 16 and transglutaminase.

Item 18
Item 18. The biotin labeling kit according to Item 17, wherein the transglutaminase is a microorganism-derived transglutaminase.

Item 19
A protein or peptide biotin labeling method comprising the following steps 1 to 3;
(1) Step 1 of mixing the biotin compound according to any one of Items 1 to 14 and the protein or peptide in the presence of transglutaminase,
(2) Step 2 where the mixture of Step 1 is subjected to transglutaminase optimal activity conditions,
(3) Step 3 of recovering biotin-labeled protein or biotin-labeled peptide from the mixture after Step 2.

Item 20
Item 20. The method according to Item 19, wherein the protein or peptide comprises one or more amino acid sequences represented by any one of SEQ ID NOs: 1 to 4.

Item 21
Item 21. The method according to Item 19 or 20, wherein the transglutaminase is a microorganism-derived transglutaminase.

Item 22
A protein or peptide variant in which one or more of the amino acid sequences shown in any one of SEQ ID NOs: 1 to 4 are inserted or substituted.

Item 23
Item 23. The protein or peptide variant according to Item 22, comprising the amino acid sequence represented by any of SEQ ID NOs: 8 to 13.

Item 24
The following method for producing a protein or peptide aggregate, comprising steps 1 to 3;
(1) Step 1 of mixing the protein or peptide variant according to Item 22 or 23, the biotin compound according to any one of Items 1 to 14, the avidin compound, and transglutaminase,
(2) Step 2 where the mixture of Step 1 is subjected to the optimal activity condition of the transglutaminase,
(3) Step 3 of recovering a protein or peptide aggregate from the mixture after Step 2.

Item 25
Item 25. The method according to Item 24, wherein the transglutaminase is a microorganism-derived transglutaminase.

Item 26
Item 26. A protein or peptide aggregate obtained by the method according to Item 24 or 25.

Hereinafter, the present invention will be described in more detail.
Various techniques used for carrying out the present invention can be easily and surely implemented by those skilled in the art based on known documents and the like, except for a technique that clearly indicates the source. For example, in the case of genetic engineering and molecular biology techniques, Sambrook and Russell, “Molecular Cloning A LABORATORY MANUAL”, Cold Spring Harbor Laboratory Press, New York, (2001); Ausubel, F .; M.M. et al. “Current Protocols in Molecular Biology”, John Wiley & Sons, New York,. References such as NY may be referred to.

Biotin Compound The biotin compound of the present invention is represented by the formula (1).

  In the formula (1), n is 1 or 2.

  In the formula (1), X is O or NH. Further, when n is 2, X may be the same or different.

In formula (1), R ¹ is a direct bond or an amino acid residue. When n is 2, the amino acid residues of R ¹ may be the same or different.

In formula (1), R ³ is a direct bond or an amino acid residue.

In the formula (1), each of the amino acid residues of R ¹ or R ^{3 is} an amino acid residue in which one or two or more amino acid residues are peptide-bonded.

  In the formula (1), an amino acid residue means that one hydrogen atom is removed from an amide group of an amino acid (this group is referred to as an amino residue in this specification), and one OH is converted from a carboxyl group. (This group is referred to herein as a carboxy residue.) Here, the amino acid is not limited to amino acids present in the living body, and is a compound having an amide group and a carboxyl group.

  The above amino acid includes proline. That is, when the amino acid residue is a proline residue, it does not have the amino residue in which one hydrogen atom is removed from the amino group described above, but one hydrogen atom is removed from the imino group in the pyrrolidine ring structure. Have

  In addition, an amino acid residue in which two or more amino acids are peptide-bonded means that one hydrogen atom is removed from the N-terminal amide group of the main chain of the protein or peptide in which the above-mentioned amino acid residues are peptide-bonded, and C One OH is removed from the terminal carboxyl group. These groups are also referred to herein as amino and carboxy residues, respectively.

In Formula (1), when R ¹ is an amino acid residue, the amino residue of the amino acid residue and a (C═O) group adjacent to R ¹ are peptide-bonded.

In the formula (1), when n is 1, R ² is a direct bond. In this case, when R ¹ is an amino acid residue and R ³ is an amino acid residue, the carboxylic acid residue of the amino acid residue of R ¹ and the carboxylic acid residue of the amino residue of R ³ The group is peptide bonded. When R ³ is a direct bond, the carboxylic acid residue of the amino acid residue of R ¹ and the (NH) group adjacent to R ³ are peptide-bonded.

In Formula (1), when n is 1, R ¹ is a direct bond, and R ³ is an amino acid residue, a group (C═O) adjacent to R ¹ and R ³ Of the amino acid residues are peptide-bonded.

In the formula (1), when n is 2, R ² is an amino acid residue having an amino residue obtained by removing one hydrogen atom from the terminal amino group of the side chain.

Here, when R ¹ is an amino acid residue, an amino residue obtained by removing one hydrogen atom from the amino group at the end of the side chain and a carboxylic acid residue of the amino acid residue of R ¹ are peptide bonds. doing. In addition to such a peptide bond, a peptide bond is formed between the carboxylic acid residue in the amino acid residue of R ^{1 and} the amino residue defined by the amino acid residue of R ² .

In the case where R ¹ is a direct bond, the amino residue in which one hydrogen atom is removed from the amino group at the end of the side chain is also an amino residue defined by the amino acid residue of R ² Are also bonded to the (C═O) group adjacent to R ¹ .

That is, in the above formula (1), when n is 2, R ² is peptide-bonded to R ¹ at two positions.

In the formula (1), when n is 2, the carboxylic acid residue of R ² is peptide-bonded to the amino residue of the amino acid residue of R ³ . When R ³ is a direct bond, the carboxylic acid residue of R ² and the (NH) group adjacent to R ³ are peptide-bonded.

In the formula (1), the R ² when n is 2 is not particularly limited, but is preferably a lysine residue.

In formula (1), the amino acid residue of R ¹ or R ³ is not particularly limited, and examples thereof include an amino acid residue containing at least one selected from the group consisting of a glycine residue or a leucine residue. .

When R ¹ is an amino acid residue in which two or more amino acid residues are peptide-bonded, the upper limit on the number is not limited, but for example, an amino acid residue in which six or less amino acid residues are peptide-bonded Based on it. More preferably, it is 5 or less, more preferably 4 or less, and most preferably 3 or less.

When R ³ is an amino acid residue in which two or more amino acid residues are peptide-bonded, the upper limit on the number is not limited, but for example, an amino acid residue in which six or less amino acid residues are peptide-bonded Based on it. More preferably, it is 5 or less, more preferably 4 or less, and most preferably 3 or less.

Among the biotin compounds represented by formula (1), one of the most preferred biotin compounds is a biotin compound represented by formula (4). The biotin compound represented by the formula (4) is a biotin compound in which n is 1 and R ¹ and R ³ are a direct bond in the formula (1). Hereinafter, in this specification, the biotin compound represented by the formula (4) may be referred to as biotin-QG.

Another embodiment of the biotin compound represented by the formula (1) is a biotin compound represented by the formula (2).

Formula (2) is a biotin compound in which R ³ is an amino acid residue in which two or more amino acid residues are peptide-bonded in Formula (1), and contains a leucine residue on the carboxy terminal side of the amino acid residue It is.

  In the formula (2), n is 1 or 2.

  In the formula (2), X is O or NH. Further, when n is 2, X may be the same or different.

In formula (2), R ¹ is a direct bond or an amino acid residue. When n is 2, the amino acid residues of R ¹ may be the same or different.

In formula (2), R ⁴ is a direct bond or an amino acid residue.

In formula (2), the amino acid residue of R ¹ or R ^{4 is} an amino acid residue in which one or two or more amino acid residues are peptide-bonded.

  The amino acid residue and the amino acid residue in which two or more amino acids are peptide-bonded in the formula (2) are the same as those in the formula (1).

In Formula (2), when R ¹ is an amino acid residue, the amino residue of the amino acid residue and a (C═O) group adjacent to R ¹ are peptide-bonded.

In the formula (2), when n is 1, R ² is a direct bond. In this case, when R ¹ is an amino acid residue and R ⁴ is an amino acid residue, the carboxylic acid residue of the amino acid residue of R ¹ and the carboxylic acid residue of the amino residue of R ⁴ The group is peptide bonded. Further, when R ⁴ is a direct bond, the carboxylic acid residue of the amino acid residue of R ¹ and the (NH) group adjacent to R ⁴ are peptide-bonded.

In Formula (2), when n is 1, R ¹ is a direct bond, and R ⁴ is an amino acid residue, a group adjacent to R ¹ (C═O) and R ⁴ Of the amino acid residues are peptide-bonded.

In the formula (2), R ² when n is 2 is an amino acid residue having an amino residue obtained by removing one hydrogen atom from the amino group at the end of the side chain.

Here, when R ¹ is an amino acid residue, an amino residue obtained by removing one hydrogen atom from the amino group at the end of the side chain and a carboxylic acid residue of the amino acid residue of R ¹ are peptide bonds. doing. In addition to such a peptide bond, a peptide bond is formed between the carboxylic acid residue in the amino acid residue of R ^{1 and} the amino residue defined by the amino acid residue of R ² .

In the case where R ¹ is a direct bond, the amino residue in which one hydrogen atom is removed from the amino group at the end of the side chain is also an amino residue defined by the amino acid residue of R ² Are also bonded to the (C═O) group adjacent to R ¹ .

That is, in the above formula (2), when n is 2, R ² is peptide-bonded to R ¹ at two positions.

In the formula (2), when n is 2, the carboxylic acid residue of R ² is peptide-bonded to the amino residue of the amino acid residue of R ⁴ . When R ⁴ is a direct bond, the carboxylic acid residue of R ² and the (NH) group adjacent to R ⁴ are peptide-bonded.

In the formula (2), R ² when n is 2 is not particularly limited, but is preferably a lysine residue.

In the formula (2), the amino acid residue of R ¹ or R ⁴ is not particularly limited, and examples thereof include an amino acid residue containing at least one glycine residue.

When R ¹ is an amino acid residue in which two or more amino acid residues are peptide-bonded, the upper limit on the number is not limited, but for example, an amino acid residue in which six or less amino acid residues are peptide-bonded Based on it. More preferably, it is 5 or less, more preferably 4 or less, and most preferably 3 or less.

When R ⁴ is an amino acid residue in which two or more amino acid residues are peptide-bonded, the upper limit on the number is not limited, but for example, an amino acid residue in which five or less amino acid residues are peptide-bonded Based on it. More preferably, it is 4 or less, most preferably 3 or less.

Among the biotin compounds represented by formula (2), one of the most preferred biotin compounds is a biotin compound represented by formula (5). The biotin compound represented by the formula (5) is a biotin compound in which n is 1 and R ⁴ is an amino acid residue in which 6 glycine residues are peptide-bonded in the formula (2) It is. Hereinafter, in this specification, the biotin compound represented by the formula (5) may be referred to as biotin-GGG-GGLQG.

Another embodiment of the biotin compound represented by the formula (1) is a biotin compound represented by the formula (3).

In formula (3), in formula (1), n is 2, R ¹ corresponds to R ⁵ and R ⁶ respectively, R ² is a lysine residue, and R ³ is 2 or more amino acids It is an amino acid residue in which the residue is a peptide bond, and is a biotin compound containing a leucine residue on the carboxy terminal side of the amino acid residue.

Further, the amino acid residue in which one hydrogen atom from the amino group of ε-position end of the side chain is removed lysine residues, corresponding to R ⁵ in the formula (3), a carboxylic of R ¹ of formula (1) It is peptide-bonded to an acid residue and a peptide, and to an amino residue of a lysine residue and a carboxylic acid residue of R ¹ corresponding to R ⁶ in formula (3).

In addition, when R ⁵ or R ⁶ is a direct bond, it is peptide-bonded to the (C═O) group adjacent to R ¹ in formula (1).

  In the formula (3), X is O or NH, and these may be the same or different.

In Formula (3), R ⁵ , R ⁶ , or R ⁷ is a direct bond or an amino acid residue.

In formula (3), each of R ⁵ , R ⁶ , or R ^{7 is} an amino acid residue in which one or more amino acid residues are peptide-bonded.

  The amino acid residue in Formula (3) and the amino acid residue in which two or more amino acids are peptide-bonded are the same as those in Formula (1) or Formula (2).

In the formula (3), when R ⁵ is an amino acid residue, the amino residue of the amino acid residue and the (C═O) group adjacent to R ⁵ are peptide-bonded, and A peptide bond is formed between the acid residue and an amino residue obtained by removing one hydrogen atom from the ε-terminal amino group of the side chain of the lysine residue.

In Formula (3), when R ⁵ is a direct bond, one hydrogen atom was removed from the (C═O) group adjacent to R ⁵ and the ε-terminal amino group of the side chain of the lysine residue. Amino residues are peptide-bonded.

In Formula (3), when R ⁶ is an amino acid residue, the amino residue of the amino acid residue and the (C═O) group adjacent to R ⁶ are peptide-bonded, and A peptide bond is formed between the carboxylic acid residue and the lysine residue amino residue.

In Formula (3), when R ⁶ is a direct bond, the (C═O) group adjacent to R ⁶ and the amino residue of the lysine residue are peptide-bonded.

In the formula (3), when R ⁷ is an amino acid residue, the amino residue of the amino acid residue and the carboxylic acid residue of the lysine residue are peptide-bonded, and the carboxylic acid of the amino acid residue and residues, adjacent to ^{R 7} are bonded (NH) group and a peptide.

When R ⁷ is a direct bond, the amino residue of the lysine residue and the (C═O) group adjacent to R ⁷ are peptide-bonded.

The amino acid residues in R ⁵ _, R ⁶ , and R ⁷ are not particularly limited, and examples include amino acid residues including at least one glycine residue.

When R ⁵ or R ⁶ is an amino acid residue in which two or more amino acid residues are peptide-bonded, the upper limit on the number is not limited, but for example, six or less amino acid residues are peptide A bonded amino acid residue may be used. More preferably, it is 5 or less, more preferably 4 or less, and most preferably 3 or less.

When R ⁷ is an amino acid residue in which two or more amino acid residues are peptide-bonded, the upper limit on the number is not limited, but for example, an amino acid in which five or less amino acid residues are peptide-bonded It may be a residue. More preferably, it is 4 or less, and more preferably 3 or less.

Among the biotin compounds represented by formula (3), the most preferred biotin compound is a biotin compound represented by formula (6). In the biotin compound represented by formula (6), in formula (3), R ⁵ and R ⁶ are amino acid residues in which three glycine residues are peptide-bonded, and one R ⁷ is present. It is a biotin compound that is a glycine residue. Hereinafter, in this specification, the biotin compound represented by the formula (6) may be referred to as bis- (biotin-GGG) -KGLQG.

  The biotin compound of the present invention may be produced by using biotin and amino acids based on various constituent amino acid residues as raw materials and employing a general Fmoc peptide synthesis method such as solid phase synthesis. A specific peptide synthesis method includes a method according to the following procedure.

1) Assemble the PD-10 column and add 0.1 mmole of Fmoc-Gly-Alkoresin into it.

2) Shake overnight with 3 mL DMF to swell the resin.

3) Wash 3 times with 3 mL DMF to remove stabilizer.

4) Wash once with 3 mL of 20% PPD / DMF to deprotect Fmoc.

5) Shake with 3 mL of 20% PPD / DMF for 15 minutes to deprotect Fmoc.

6) Wash 3 times with 3 mL DMF.

7) Add 0.5 mmol Fmoc-various amino acids and 0.45 M HBTU, 2.1 mL HOBt / DMF solution, 0.7 mL 0.9 M DIEA / DMF solution and shake for an appropriate time.

8) Wash 3 times with 3 mL DMF. Here, the unreacted resin may be confirmed by a Kaiser test. If the test result is positive, the process may return to 7) and the amino acid may be re-extended. If the test result is negative, the following operation can be performed.

9) Wash 3 times with 3 mL DCM.

10) Wash 3 times with 3 mL DMF.

11) Wash once with 3 mL of 20% PPD / DMF to deprotect Fmoc.

12) Shake with 3 mL of 20% PPD / DMF for 15 minutes to deprotect Fmoc.

13) Wash 3 times with 3 mL DMF.

14) The above 7) to 13) are repeated, and the amino acids constituting it are extended so as to be the target biotin compound. For example, a compound such as Fmoc-Lys (Fmoc) -OH or Fmoc-Gly is appropriately employed and extended.

15) Add biotin or iminobiotin, 0.45 M HBTU, 2.1 mL HOBt / DMF solution, 0.7 mL 0.9 M DIEA / DMF solution to the amino acid-extended resin, and shake at an appropriate time. To do.

16) Wash 5 times with 3 mL DCM, 5 times with 3 mL DCM, 5 times with 3 mL methanol and then vacuum dry overnight.

17) Add 2.5 mL cocktail of TFA, TIS, and deionized water mixed at 95: 2.5: 2.5, respectively, and stir for 1 hour to cut out from the resin.

18) 40 mL of diethyl ether is added to the solution cut out after all the elongation operations to precipitate, and a crude peptide is obtained by lyophilization.

19) The obtained crude peptide is purified by HPLC by tracking the absorption at 230 nm derived from the peptide under the following conditions, and the fraction corresponding to the product is collected and lyophilized. The HPLC conditions are shown in Table 1.

  Table 2 shows an example of the analysis result by the MS spectrum of the biotin compound represented by the above formulas (4), (5), and (6).

Biotin Labeling Agent The biotin compound of the present invention is particularly useful when biotinylating amino acids or proteins with other reagents or as they are. Therefore, in the present invention, a biotin labeling agent for a protein or peptide containing the above-described biotin compound is provided.

  In addition to the biotin compound of the present invention, biotin compounds such as N-BIOTINYL-3, 6, 9-TRIOXAUNDECANENE-1, 11-DIAMINE and biotin cadaverine are also useful as the biotin labeling agent of the present invention. These can be obtained from Molecular Biosciences.

  In addition to the biotin compound of the present invention described above, the biotin labeling agent of the present invention may contain a known substance that is usually contained in a reagent as long as biotinylation to an amino acid or protein is not hindered. In such a biotinylated labeling agent, the above biotin compound may be contained in an amount of 0.001 to 99.9 parts by weight.

Although the protein or peptide which is the target of the biotin labeling agent of the present invention is not particularly limited, for example, it preferably contains any one or more of the amino acid sequences shown in SEQ ID NOs: 1 to 4. Among these amino acid sequences, the presence and position of proline residues and lysine residues are important, and more preferably, any one or more of the amino acid sequences shown in SEQ ID NOs: 5 to 7.

  Such an amino acid sequence may be contained at the N-terminal or C-terminal of the protein or peptide, or may be contained at other sites. Moreover, it is preferable that the amino acid sequence is contained in the site | part of the position close | similar to the outer side instead of the inside in the three-dimensional structure of protein or peptide.

  For example, when the protein is alkaline phosphatase, the 219th lysine residue and the 221st glutamine residue are included when inserted instead of the site between the 91st lysine residue and the 93rd threonine residue. The case where it inserts instead of the site | part between and is included is mentioned.

  Moreover, when protein is EGFP, the case where it is inserted and contained in C terminal is mentioned.

  The biotin modifying agent for the protein or peptide of the present invention is preferably used together with transglutaminase which is a kind of transferase. The transglutaminase is not particularly limited as long as it is an enzyme that catalyzes an acyl transfer reaction between glutamine and a compound having lysine or a primary amine group. For example, a transglutaminase derived from a microorganism is preferable. More specifically, it is a transglutaminase derived from Streptomyces mobaraensis, NCBI Accession No. It is a transglutaminase having the amino acid sequence shown by Q8KRJ2P81453.

  The present invention also provides a biotin labeling kit comprising a biotin modifying agent for the above protein or peptide and the above transglutaminase.

  The biotin labeling kit of the present invention may further contain known components, reagents and the like.

Protein or Peptide Variant of the Present Invention A variant of the present invention is a protein or peptide variant in which one or more of the amino acid sequences shown in any of SEQ ID NOs: 1 to 4 are inserted or substituted.

  The insertion may be inserted at the N-terminus, C-terminus, or other site of the protein or peptide.

  The substitution may be substituted with the amino acid sequence at the N-terminal, C-terminal, or other site of the protein or peptide.

  For example, when the protein or peptide is alkaline phosphatase, the 219th lysine residue and the 221st glutamine are inserted or substituted in place of the site between the 91st lysine residue and the 93rd threonine residue. The case where it inserts or substitutes instead of the site | part between residues is mentioned.

  In addition, if the protein or peptide is EGFP, it may be inserted or substituted in place of the C-terminal 5 amino acid site.

Examples of the protein or peptide variant of the present invention include:
Examples include protein variants or peptides containing the amino acid sequences shown in SEQ ID NOs: 8 to 13.

  The method for producing the protein or peptide variant of the present invention is not particularly limited, and a known biochemical method may be used. For example, a nucleic acid encoding the protein variant or peptide of the present invention is produced, an appropriate host is transformed using the nucleic acid, biosynthesized by culturing the host, and finally obtained after the culture. The protein variant or peptide may be recovered from the host and purified as necessary.

  Such a protein or peptide variant may further be mutated as long as the function of the protein or peptide variant is not impaired. The homology between such a mutant and the mutant of the present invention is about 85%, more preferably about 90%, still more preferably about 95%, and most preferably about 99%. Homology is also called identity.

  Amino acid sequence identity refers to the degree of two or more comparable amino acid sequences or base sequences that are identical to each other. Therefore, the higher the identity of a certain two amino acid sequences or base sequences, the higher the identity or similarity of those sequences. The level of amino acid sequence or base sequence identity is determined, for example, using FASTA, a sequence analysis tool, using default parameters.

  Alternatively, when analyzing an amino acid sequence, BLASTX may be used, and as parameters, for example, score = 50 and wordlength = 3 may be used.

When using BLAST and Gapped BLAST programs, the default parameters of each program may be used. Specific methods of these analysis methods are known, and the National Center of Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/)
Please refer to.

  In addition, the protein or peptide variant according to the present invention may have a known tag sequence as long as the effects of the present invention described below are not impaired. The tag sequence may have, for example, any of the amino acid sequence of the protein or peptide variant, at the N-terminus, or at the C-terminus.

  The protein or peptide variant according to the present invention produces a nucleic acid containing a base sequence encoding it based on an amino acid sequence, and uses such nucleic acid as a host suitable for protein synthesis such as Escherichia coli, yeast, insect cells, mammalian cells, etc. It can be produced by introducing into cells. A specific method is not particularly limited, and a known method may be used.

  The protein or peptide variant according to the present invention may be produced through a purification step as appropriate. The specific production method is not particularly limited, but may be purified using known means such as column chromatography, acetone precipitation method, ammonium sulfate precipitation method and the like.

  Specific column chromatography includes ion exchange column chromatography, affinity column chromatography, reverse phase column chromatography, hydrophobic column chromatography, gel filtration chromatography, and the like, one or two of these. What is necessary is just to employ | adopt combining the above.

Biotin Labeling Method of the Present Invention The biotin labeling method of the present invention is a method for labeling a protein or peptide with biotin, that is, a method for biotinylating a protein or peptide, and includes the following steps 1 to 3.

<Step 1>
Step 1 of mixing the above-described biotin compound of the present invention and a protein or peptide in the presence of transglutaminase.

<Step 2>
Step 2 where the mixture of Step 1 is placed under optimal activity conditions for transglutaminase.

<Step 3>
Step 3 of recovering biotin-labeled protein or biotin-labeled peptide from the mixture after Step 2.

Step 1 Step 1 in the method for labeling a protein or peptide of the present invention with biotin is a step of mixing the above-described biotin compound of the present invention with a protein or peptide in the presence of transglutaminase.

  Step 1 may be mixed in an appropriate solvent, and the kind thereof is not particularly limited. For example, a well-known buffer frequently used in biochemical experiments may be used. For example, 20 mM Tris-HCl (pH 8. 0) and the like.

  The protein or peptide may be the same as the amino acid or protein targeted by the biotin labeling agent of the present invention described above. Such a protein or peptide is obtained by the above-described method for producing a protein or peptide variant of the present invention.

  The transglutaminase is not particularly limited, and for example, transglutaminase used together with the above-described biotin modifier for the protein or peptide of the present invention may be employed.

  The amount of transglutaminase used may be such that the final concentration in the mixture is usually about 0.01 to 1 U / ml.

  In addition, the biotin compound and the protein or peptide may be mixed in such an amount that the molar ratio is usually about 1: 1 to 1:20.

Step 2 Step 2 in the protein or peptide biotin labeling method of the present invention is a step of placing the mixture of Step 1 under the optimal activity condition of transglutaminase.

  As described above, since the transglutaminase used in Step 1 is an enzyme that catalyzes an acyl transfer reaction, the condition in Step 2 is that the reaction proceeds smoothly.

  Specifically, the temperature may be about 4 to 50 ° C. and the pH may be about 5 to 9. The reaction time is not particularly limited, but may be about 5 minutes to 24 hours. This reaction time is the time for “putting under optimum activity conditions” in step 2.

Step 3 Step 3 in the biotin labeling method of protein or peptide of the present invention is a step of recovering biotin-labeled protein or biotin-labeled peptide from the mixture after step 2. This step may include a step of purifying the biotin-labeled protein or biotin-labeled peptide as necessary.

  The specific production method is not particularly limited as long as it is a means for removing the biotin compound of the present invention and the unmodified protein or peptide from the mixture obtained in step 2. For example, a purification method employed in the production of the above-described protein or peptide variant of the present invention or a method obtained by appropriately modifying this method can be mentioned.

Protein or peptide assembly The protein or peptide assembly according to the present invention is produced by a method including the following steps 1 to 3.

≪Process 1≫
Mixing protein or peptide variant, biotin compound, avidin compound and transglutaminase 1,

≪Process 2≫
Step 2 wherein the mixture of Step 1 is subjected to the optimal activity condition of the transglutaminase.

≪Process 3≫
Step 3 for recovering the alkaline phosphatase aggregate after Step 3 above.

Step 1 Step 1 in the method for producing a protein or peptide assembly of the present invention is a step of mixing a protein or peptide, a biotin compound, an avidin compound, and transglutaminase.

  The protein or peptide used in step 1 may be the same as that used in step 1 of the biotin labeling method of the present invention.

  The biotin compound used in step 1 may be the same as the biotin labeling method of the present invention described above.

  Examples of the avidin compound used in Step 1 include avidin, streptavidin, neutravidin, and the like, and although not particularly limited, strept from the viewpoint of interaction with the target protein, electrostatic interaction due to difference in isoelectric point, and the like. Avidin is preferably used.

  The transglutaminase used in step 1 may be the same as that used in step 1 of the biotin labeling method of the present invention described above.

  The specific purification method is not particularly limited, but is not particularly limited as long as it is a means for removing biotin compound, transglutaminase, biotin compound, and unmodified protein or peptide from the mixture. What is necessary is just to carry out similarly to the manufacturing method of the protein or peptide variant which concerns on this invention mentioned above.

  In addition, when a protein or peptide, a biotin compound, and transglutaminase are mixed in advance, it is preferable to put them under conditions for optimal activity of transglutaminase after mixing. Specific conditions may be the same as those described in step 2.

  The mixing amount of the protein or peptide, the biotin compound, and the avidin compound in step 1 is usually about a protein or peptide: biotin compound = 1: 1 to 1:20 in a molar ratio.

  Further, when it is assumed that the process 1 includes the formation of the complex, the biotin compound: avidin compound = 1: 0.1 to 1: 1 may be used.

  Furthermore, when the biotin compound has two biotinyl groups, the biotin compound: avidin compound may be about 1: 0.1 to 1: 2.

  The amount of transglutaminase mixed in step 1 is not particularly limited, but the final concentration in the mixture may be about 0.01 to 1 U / ml.

  In step 1, the protein or peptide, biotin compound, avidin compound, and transglutaminase may be mixed in a suitable solvent. Although a specific solvent is not particularly limited, a known buffer used in biochemical experiments may be used.

Step 2 Step 2 is a step in which the mixture shown in Step 1 is subjected to the optimal activity condition of the above-mentioned transglutaminase. As described above, since the transglutaminase used in Step 1 is an enzyme that catalyzes an acyl transfer reaction, the condition in Step 2 is that the reaction proceeds smoothly.

  Specifically, the temperature may be about 4 to 50 ° C. and the pH may be about 5 to 9. The reaction time is not particularly limited, but may be about 5 minutes to 24 hours. This reaction time is the time for “putting under optimum activity conditions” in step 2.

About Step 3 Step 3 is a step of recovering the protein or peptide aggregate after Step 2. In this step, the recovery may include a purification step.

  The specific purification method is not particularly limited as long as it is a means for removing the protein or peptide variant, biotin compound, transglutaminase, and biotin compound from the mixture obtained in step 2. There is no particular limitation, and the method may be the same as the above-described method for producing a protein or peptide variant according to the present invention.

  Hereinafter, the case where the protein or peptide of the present invention is an alkaline phosphatase aggregate will be described in detail.

  Alkaline phosphatase aggregates form tetramers or higher. And since the alkaline phosphatase which is a raw material which forms an aggregate forms a dimer, an alkaline phosphatase aggregate is an even number of monomers such as a tetramer, a hexamer, an octamer, etc. Preferably formed by the body.

  The molecular diameter of the alkaline phosphatase aggregate is not particularly limited, but is usually preferably 13 nm or more. Moreover, although molecular weight is not specifically limited, Usually, 200 kDa or more is preferable.

  The effects of the biotin compound of the present invention are listed below. Needless to say, the biotin compound of the present invention is not limited to those having all the following effects.

  The biotin compound of the present invention can specifically bind to a protein or peptide having a specific amino acid sequence by using it together with transglutaminase. Therefore, the biotin compound of the present invention is suitable as a biotin modifying agent.

  The effects of the protein or peptide variant of the present invention are listed below. In addition, it cannot be overemphasized that the protein or peptide variant which concerns on this invention is not limited to what has all the following effects.

  The protein or peptide variant according to the present invention has a sequence recognized by transglutaminase and can specifically modify amino acids in the vicinity of such a sequence.

  The enzyme activity of the protein or peptide variant that has been modified as described above is not significantly reduced compared to the wild-type protein or peptide, and also exhibits the enzyme activity while achieving the purpose of the modification. Therefore, it can be a multifunctional molecule.

  Since the protein or peptide variant of the present invention can be modified so as not to affect the three-dimensional structure of the wild type, it can be aggregated, and such aggregated protein or peptide Can be an assembly having a remarkably excellent function.

The figure which shows the FITC modification experiment result of various alkaline phosphatase variants. (A) Experimental conditions of various alkaline phosphatase mutants modified with FITC. (B) Images subjected to CBB staining after SDS-PAGE of various FITC-modified alkaline phosphatase mutants. (C) Fluorescence photographic images after SDS-PAGE of various FITC-modified alkaline phosphatase mutants. The figure which shows the FITC modification experiment result of various alkaline phosphatase variants. (A) Experimental conditions of various alkaline phosphatase mutants modified with FITC. (B) Images subjected to CBB staining after SDS-PAGE of various FITC-modified alkaline phosphatase mutants. (C) Fluorescence photographic images after SDS-PAGE of various FITC-modified alkaline phosphatase mutants. The figure which shows the FITC modification experiment result of various alkaline phosphatase variants. (A) Experimental conditions of various alkaline phosphatase mutants modified with FITC. (B) Images subjected to CBB staining after SDS-PAGE of various FITC-modified alkaline phosphatase mutants. (C) Fluorescence photographic images after SDS-PAGE of various FITC-modified alkaline phosphatase mutants. The figure which shows the biotin modification experiment result of AP (219-221) -K2 variant. (A) CBB stained image when biotin-QC is used. (B) CBB stained image when biotin-GGG-GGLQG is used. (C) CBB stained image using bis (biotin-GGG) -KGLQG. The figure which shows the biotin modification experiment result of AP (219-221) -Q variant. (A) Experimental conditions. (B) CBB stained image. The figure which shows the DLS experiment result of the composite_body | complex of a biotin modification alkaline phosphatase variant (AP (219-221) -K2). (A) When alkaline phosphatase modified with biotin-QG is used. (B) When alkaline phosphatase modified with biotin-GGG-GGLQG is used. (C) When alkaline phosphatase modified with bis (biotin-GGG) -KGLQG is used. The figure which shows the molecular exclusion chromatography experiment result of the aggregate | assembly of a biotin modification alkaline phosphatase variant (AP (219-221) -K2). (A) When alkaline phosphatase modified with biotin-QG is used. (B) When alkaline phosphatase modified with biotin-GGG-GGLQG is used. (C) When alkaline phosphatase modified with bis (biotin-GGG) -KGLQG is used. The figure which shows the complex formation experiment result of an alkaline phosphatase aggregate (219-221) -Q. (A) The figure which shows a DLS experiment result. (B) The figure which shows a molecular exclusion chromatography experiment result. The schematic diagram which shows the shape of an alkaline phosphatase aggregate. The experimental result which confirmed the function of the alkaline phosphatase complex by indirect ELISA. (A) When AP (219-221) -K2 mutant is used. (B) When AP (219-221) -Q mutant is used. (C) When chemically modified biotinylated AP is used. The figure which shows the formation experiment result of the alkaline phosphatase aggregate on a streptavidin coating plate. The figure which shows the formation experiment result of an EGFP aggregate | assembly.

  Examples for explaining the present invention in more detail are described below. In addition, it cannot be overemphasized that this invention is not limited to the content as described in the Example shown below.

<Example>
Experimental Example 1: Preparation of alkaline phosphatase mutant The alkaline phosphatase mutant shown in Table 3 below was prepared. Specifically, a base sequence encoding various alkaline phosphatase mutants in which an amino acid sequence consisting of MDIGINSDPHHHHH (NHis-AP: SEQ ID NO: 14) is added to the N-terminus of the horn mutant is used as an E. coli expression vector (pET22b ( An alkaline phosphatase mutant expression vector incorporated in (+)) was prepared. E. coli BL21 (DE3) strain was transformed with such a vector.

  The transformant was cultivated in an appropriate medium and then collected, and then subjected to a crushing step to obtain a cell lysate of the transformant. Thereafter, various alkaline phosphatase mutants were isolated and purified from the cell lysate according to a conventional method. In the purification process, a Ni-NTA column was used.

Experimental Example 2: Activity measurement of various alkaline phosphatase mutants The activities of various alkaline phosphatases prepared in Experimental Example 1 were measured. This is for confirming whether or not the activity is affected by the mutation. As a substrate for alkaline phosphatase, p-nitrophenyl phosphate (p-NPP) was used. The activity was measured at 25 ° C., and 10 μl of various alkaline phosphatase solutions (concentration 2 μM) were added to 990 μl of 1 mM p-NPP / 1M Tris-HCl (pH 8.0) solution, and the absorbance at 410 nm was traced. It was. The results are shown in Table 4.

  As is clear from Table 4, it was revealed that the activities of the various alkaline phosphatase mutants prepared in Experimental Example 1 were hardly different from the wild type. Therefore, it can be said that the mutation introduction hardly affects the activity of alkaline phosphatase itself.

Experimental Example 3: FITC modification of various alkaline phosphatase mutants Among the various alkaline phosphatase mutants prepared in Experimental Example 1, E219K, W220K, Q221K, AP (91-93) -K1, AP (91-93)- K2, AP (219-221) -K1, and AP (219-221) -K2 were modified with FITC-β-Ala-QG (Org. Biomol. Chem., 2009, 7, 3407-3412). This is a compound in which FITC is covalently bonded to the N-terminus of an Ala-Gln-Gly tripeptide, and is known as a substrate for transglutaminase.

  FITC- in a 25 mM Tris-HCl (pH 8.0), 137 mM NaCl, 2.68 mM KCl TBS buffer with final concentrations of 200 μM, 0.5 mg / ml (10 μM), and 0.5 U / ml, respectively. Add β-Ala-QG, the above three types of alkaline phosphatase mutants, and MTG (Org. Biomol. Chem., 2009, 7, 3407-3412), stir well, and let stand at 25 ° C for 3 hours. I went there. In addition, a comparative experiment in which MTG was not added was also performed.

Thereafter, SDS-PAGE was performed on samples at 0, 60, and 180 minutes after the start of the reaction. The fluorescence after FITC was observed for the gel after electrophoresis using a fluorescence imager.
Furthermore, CBB staining was also performed after observation. As a comparative experiment, a polypeptide (NHis) having the amino acid sequence shown in SEQ ID NO: 10 was used in place of the six types of alkaline phosphatase mutants. These results are shown in FIGS. 1-1 and 1-2.

  In addition, the alkaline phosphatase mutant AP (219-221) -Q produced in Experimental Example 1 is a primary amine, Fluorescein cadaverine ([5-((5-Aminopentyl) thioureidyl) fluorescein]: AnaSpec) Modified with This is known as a substrate for transglutaminase.

  Fluorescein cadaverine in 25 mM Tris-HCl (pH 8.0), 137 mM NaCl, 2.68 mM KCl TBS buffer to final concentrations of 200 μM, 0.5 mg / ml (10 μM), and 0.5 U / ml, respectively. , AP (219-221) -Q and MTG were added, and the mixture was stirred well and allowed to stand at 25 ° C. for 3 hours. In addition, a comparative experiment in which MTG was not added was also performed.

  Then, SDS-PAGE was performed on the samples after 0, 10, 30, 60, 120, and 180 minutes after the start of the reaction. The fluorescence after FITC was observed for the gel after electrophoresis using a fluorescence imager. Furthermore, CBB staining was also performed after observation. These results are shown in FIGS. 1-3.

  Furthermore, the activity of alkaline phosphatase mutants to which the above seven types of FITC modifications were applied was measured. In order to complete the modification by FITC, the FITC-modified alkaline phosphatase mutant obtained by setting the reaction time for all the mutants to 24 hours was subjected to an activity measurement experiment. A specific measurement of alkaline phosphatase activity was performed by the same method as in Experimental Example 2 above. The results are shown in Table 5.

  From FIGS. 1-1 and 1-2, various alkaline phosphatase mutants were observed at about 50 kDa, and an MTG band was observed at about 38 kDa.

  In particular, from FIG. 1-1, a slight increase in the band was observed for only W220K, that is, a mutant in which the 220th tryptophan residue of alkaline phosphatase was mutated to lysine and MTG was present. In addition, fluorescence derived from FITC was observed at the same position as the staining position with CBB. From this, it was found that even single residue mutation can be recognized by MTG.

  However, the reactivity is very low and almost unreacted alkaline phosphatase remains. Similarly, E219K and Q221K in which single residue mutations were made were not recognized by MTG, and an increase in the band and fluorescence derived from FITC were observed. I couldn't do it.

  From this result, W220K was successfully recognized by MTG, but since MTG recognition depends on the nature of amino acid residues before and after lysine residues, a new MTG recognition site is constructed by single residue mutation. Is not preferable from the viewpoint of versatility because it contains a flaky element that it is difficult to know whether it is recognized or not, although the decrease in the original functional activity of the protein is small.

  Also, from FIG. 1-2, in various alkaline phosphatase mutants into which K-loop consisting of 13 amino acid residues was inserted, a slight band shift and FITC-derived fluorescence were observed in the presence of MTG.

  Such reaction was predicted to be almost saturated with alkaline phosphatase at 20 equivalents of substrate and a reaction time of about 60 minutes. From this, it was confirmed that the inserted K-loop was recognized by the MTG as intended. Furthermore, considering the intensity of the FITC-derived fluorescence band, the RK (IRINRGPGRAFVT) sequence is more responsive than the K-loop KR (IRINRGPGRAFVT), and the insertion site 219-221 is more reactive than 91-93. It is also clear that the nature is high.

  The former is considered to be because the RK sequence was more suitable for the sequence around the lysine residue in MTG recognition. Regarding the insertion site, it is considered that the positions 219 to 221 are lower in steric hindrance than the positions 91 to 93, and the MTG is easy to approach.

  From the above two points, among the various alkaline phosphatase mutants prepared this time, AP (219-221) -K2 in which K-loop of the RK sequence is inserted at positions 219 to 221 has the highest reactivity with MTG. It can be said.

  Considering this result, until now, the insertion of sequences recognized by MTG has been mainly at the N- or C-terminal of proteins, but if K-loop of the above RK sequence is used, It can be said that the choice of the introduction site of the recognized sequence has greatly expanded.

  In addition, from FIG. 1-3, similarly to the mutant with the above K-loop inserted, AP (219-221) -Q, which is a Q-loop inserted AP mutant inserted at amino acid numbers 219-221, is also increased by MTG. It is also clear that it is labeled with efficiency.

  And from the result of Table 5, it is clear that the activity of AP (91-93) -K1 and AP (91-93) -K2 is remarkably reduced. This is presumably because the modification position of FITC for AP (91-93) -K1 and AP (91-93) -K2 was present near the active center of alkaline phosphatase. On the other hand, since AP (219-221) -K1, AP (219-221) -K2, and AP (219-221) -R maintained high activity even after modification with FITC, 219-221 was alkaline phosphatase. It can be said that it is located far from the active center.

Experimental Example 4: Biotin modification of various alkaline phosphatase mutants [AP (219-221) -K2 mutant]
Biotinylation of AP (219-221) -K2 was carried out using biotin-QG, biotin-GGG-GGLQG and bis (biotin-GGG) -KGLQG, which are biotinylated substrates of MTG.

  The enzyme reaction was carried out by adding AP (219-221) -K2, biotinylated substrate, and MTG to TBS buffer so that the final concentrations were 0.5 mg / ml (10 μM), 200 μM, and 0.5 U / ml. Was stirred well and allowed to stand at 25 ° C.

  Thereafter, the excess unreacted biotinylated substrate and biotinylated alkaline phosphatase mutant were isolated by His-tag purification using a Ni-NTA column, and the reaction was terminated. The sample after the reaction was subjected to SDS-PAGE and stained with CBB.

  Moreover, in order to confirm the degree of biotinylation, HABA assay was performed. 24.2 mg of 4-hydroxy-azobenzene-2'-carboxylic acid (HABA) was dissolved in 9.9 ml of Milli-Q water and individually stirred with 100 ml of 1N NaOH. 20 ml of egg white-derived avidin solution (10 mg) was placed in a volumetric flask, and then 600 ml of HABA solution was added. Then, the volume was adjusted with PBS to prepare a HABA-Avidin solution.

  Take 900 μl of the HABA-Avidin solution in a 1 ml cell, measure the absorbance at 500 nm, and calculate this value as Abs. A. Next, 100 μl of the above 10 μM biotinylated alkaline phosphatase mutant was placed in the cell, pipetted well, and when the absorbance was stable, the value was measured. B. The absorbance was applied to the following formula 1, and the labeling rate of biotin was calculated from the biotin concentration in the sample.

[Biotinylation rate] = 10−3 × (0.9 × Abs.A-Abs.B) / 34
(Formula 1)

  Finally, the biotinylated alkaline phosphatase was measured by two methods.

  (Activity measurement A): The same method as in Experimental Examples 1 to 3 above.

  (Activity measurement B): 0.1 μM of the above biotinylated alkaline phosphatase mutant was prepared with 25 mM Tris-HCl (pH 8.0), and 100 μL / well each of streptavidin previously layered in a 96-well microplate. And fixed at 4 ° C for 1 hour. Each well was then washed with TBST (25 mM TBS + 0.05% Tween 20). The activity of the immobilized alkaline phosphatase mutant was measured by the same method after adding the p-NPP solution to each well and reacting under the same reaction conditions. These results are shown in FIG.

  In all alkaline phosphatase mutants biotinylated with biotin-QG, biotin-GGG-GGLQG, bis (biotin-GGG) -KGLQG, a slight band shift toward high molecular weight was observed after 1 hour reaction time. It was. It is not accurate because the bands before and after modification appear to overlap, but it can be seen that the biotinylation is quite efficient. Such biotinylation can be quantitatively evaluated by the biotinylation rate measured using the HABA assay.

  Biotin-QG and biotin-GGG-GGLQG have a 100% reaction rate and a biotinylation rate of 1.00, and bis (biotin-GGG) -KGLQG has a biotinylation rate of 2.00. As a result of the measurement, almost quantitative biotinylation reaction was achieved in all the biotinylated alkaline phosphatase mutants. Therefore, it was confirmed that the newly synthesized biotinylated substrate of this time is a substrate that can be recognized by MTG and capable of quantitative biotinylation.

  Moreover, from the result of the activity measurement (activity measurement A) of the biotinylated alkaline phosphatase mutant, almost no decrease in the activity of the alkaline phosphatase mutant due to biotin modification was observed.

  Finally, in order to confirm whether the biotin group modified to the alkaline phosphatase mutant maintains the ability to bind to streptavidin, it was added and immobilized on an avidin-coated plate, and the activity in that case was also measured ( Activity measurement B).

  As a result, it was confirmed that alkaline phosphatase activity was observed on the plate, so the ability of biotin modified with alkaline phosphatase to bind to streptavidin was confirmed. At the same time, alkaline phosphatase mutants were active even when bound to streptavidin. It was confirmed that it was maintained.

[About AP (219-221) -R mutant]
Biotinylation of AP (219-221) -Q with MTG was performed using biotin- (PEO) 4-amine as a biotinylated substrate. The enzyme reaction was performed in TBS buffer so that the final concentrations were 0.5 mg / ml (10 μM), 200 μM, and 0.5 U / ml, respectively, AP (219-221) -Q, biotin- (PEO) 4− Amine and MTG were added, and they were stirred well and allowed to stand at 25 ° C.

  Subsequently, excess unreacted biotinylated substrate and biotinylated alkaline phosphatase mutant were isolated by His-tag purification using a Ni-NTA column, and the reaction was terminated. The sample after the reaction was subjected to SDS-PAGE and stained with CBB. Moreover, the biotinylation rate measurement and activity measurement were performed in the same manner as the method described in [AP (219-221) -K2 mutant]. The results are shown in Fig. 2-2.

  From the results of CBB staining after SDS-PAGE, a slight increase in the band was confirmed in the samples in the presence of MTG and biotin- (PEO) 4-amine. From the results of the activity measurement, when the enzyme activity of AP (219-221) -Q before biotinylation is 1.00, the specific activity of biotinylated AP (219-221) -Q is 0.96 ± 0.01. And succeeded in biotinylation while maintaining almost the original activity.

  Moreover, from the measurement result of the biotinylation rate using the HABA assay, the biotinylation rate of AP (219-221) -Q is 0.97 ± 0.04, and it can be said that almost quantitative biotinylation was achieved. In addition, the result of the attempt to immobilize AP (219-221) -Q on the streptavidin plate (activity measurement B) revealed that the activity of alkaline phosphatase was maintained without any problem on the plate. .

Experimental Example 5: Complex formation of biotin-modified various alkaline phosphatase mutants Various biotinylated alkaline phosphatase mutants were prepared to 2 μM by TBS after 0.2 μm filter treatment. Streptavidin solution (streptavidin biotin binding site) / (AP biotin group) (hereinafter referred to as [mSA] / [mAP]) [mSA] / [mAP] = 1/4, 1/2, In addition, the particle size of the enzyme assembly was measured using dynamic light scattering (DLS).

  The final concentration of streptavidin used was biotinylated with biotin-QG and biotin-GGG-GGLQG (219-221) -K2 and biotinylated with biotin-PEO4-amine (219-221) -Q. In the case of AP (219-221) -K2 biotinylated with bis (biotin-GGG) -KGLQG, the concentration was 4 μM.

  The solution after DLS measurement was fractionated by size exclusion column chromatography (SEC), and the behavior of the formed aggregate formation was performed according to the ratio of streptavidin and alkaline phosphatase mutant. SEC was analyzed with 20 mM TBS buffer (pH 7.4) and a flow rate of 0.5 ml / min.

  The results for various biotin-modified AP (219-221) -K2 mutants are shown in FIG. 3-1, FIG. 3-2, and Table 7.

[About biotin-QG-modified AP (219-221) -K2]
Since biotin-QG is the shortest biotinylated substrate prepared this time, it has a large steric hindrance, which prevented the self-cyclization reaction of the aggregate and expected the growth of the aggregate. As a result of DLS measurement, the particle diameter increased with the addition of streptavidin, and aggregates having a diameter of 50.3 nm were observed when [mSA] / [mAP] = 1. It is expected that the growth of the aggregate occurred as shown in the schematic diagram of FIG.

In the case of NQ-AP-CQ prepared at both ends, the maximum diameter was 31.9 nm (Y. Mori, K. Minamihata, H. Abe, M. Goto, N. Kamiya, 2011, Org. Biomol. Chem., 9, 5641.), this time, we succeeded in forming an assembly larger than that.

  Thus, (i) preparation of a biotin group as a cross-linking point on the diagonal of alkaline phosphatase, and (ii) suppression of the self-cyclization reaction with a short linker length succeeded in forming a large aggregate. .

  Next, when the sample after DLS measurement was examined by SEC, a peak derived from biotin-QG-modified AP (219-221) -K2 mutant was observed at an elution volume of 12.3 mL. This peak decreased with the addition of streptavidin and instead formed higher molecular weight aggregates.

  The peak with an elution volume of 9.8 mL is estimated to have a molecular weight of 440 kDa. From the ratio of biotin-QG-modified AP (219-221) -K2 mutant and streptavidin present in the system, there are four binding sites for streptavidin. On the other hand, biotin-QG modified AP (219-221) -K2 mutant seems to be bound respectively.

  Further, the 8.4 mL peak on both sides of the polymer was considered to be one in which one streptavidin and three biotin-QG-modified AP (219-221) -K2 mutants were further bound to the previous assembly. In addition, when [mSA] / [mAP] = 1, a large aggregate was formed so as to come out with the size exclusion volume of the column used this time. As measured by DLS, a huge aggregate was successfully formed this time. ing.

  From this, it was expected that due to the steric hindrance / stiffness of the monomer, the aggregate particle size was large, but the flexibility was poor, it could not be packed tightly, and the density was low.

[Biotin-GGG-GGLQG modified AP (219-221) -K2 mutant]
Next, biotin-GGG-GGLQG examined as a biotinylated substrate is a linear biotinylated substrate similar to biotin-QG, but the linker length is about twice that of biotin-QG. It was expected that the longer the linker, the easier the self-cyclization reaction occurs compared to biotin-QG. When the particle size was measured by DLS, the particle size increased with the addition of streptavidin, but the particle size of [mSA] / [mAP] = 1/2 was almost unchanged, and an aggregate having a diameter of about 29.2 nm Stayed formed.

  That is, as compared with the result of the biotin-QG modified AP (219-221) -K2 mutant, a decrease in the particle size of the aggregate was observed with an increase in the linker length.

  Next, when the analysis by SEC was performed, until [mSA] / [mAP] = 1/2, almost the same behavior as the biotin-QG modified AP (219-221) -K2 mutant was exhibited, and the formed aggregate was also eluted. The same volume suggested that the same aggregate was formed. However, the formation of large aggregates observed in [mSA] / [mAP] = 1 was not confirmed, and the above-mentioned two aggregates were only present in different ratios. From this, also in the analysis by SEC, the behavior that the self-cyclization reaction was promoted by the linker length was confirmed.

[Bis (biotin-GGG) -KGLQG-modified AP (219-221) -K2 mutant]
Finally, a bis (biotin-GGG) -KGLQG modified AP (219-221) -K2 mutant was combined with streptavidin to form an aggregate. The particle diameter of the aggregate increased stepwise by the addition of streptavidin, and finally became an aggregate with a diameter of 34.8 nm. The state of particle size increase due to the addition of streptavidin was clearly different from the former two, and it was expected to grow in stages. Since this is a biotin substrate having a bifurcated structure, it captures two biotin binding sites that are paired with streptavidin at the same time. -221) It was considered that the aggregate of the -K2 mutant was growing.

  When [mSA] / [mAP] = 1, the DLS measurement result was broadened in the direction of large particle diameter, so the portion where the peak was extended suggested the formation of a wire-like aggregate.

  When further analysis was performed by SEC, it was apparent that an aggregate different from the former two was formed. First, new peaks were observed at elution volumes of 10.5 mL and 11.1 mL when [mSA] / [mAP] = 1/2. These have molecular weights of 260 kDa and 320 kDa, respectively. For example, bis (biotin-GGG) -KGLQG modified AP (219-221) -K2 mutant-streptavidin- as shown in the schematic diagram of FIG. Bis (biotin-GGG) -KGLQG modified AP (219-221) -K2 mutant aggregate (AP-SA-AP aggregate) and ring self-cyclized (bis (biotin-GGG) -KGLQG modification AP (219-221) -K2 mutant-streptavidin) 2 aggregate ((AP-SA) 2 aggregate) was expected.

  In [mSA] / [mAP] = 1, the above AP-SA-AP aggregate is formed as the main, and in addition to the above (AP-SA) 2 aggregate, a new elution volume of 9.7 mL and an estimated molecular weight of 419 kDa The peak of was confirmed. Considering the molecular weight, it was considered that the (AP-SA) 2 aggregate further contained a bis (biotin-GGG) -KGLQG-modified AP (219-221) -K2 mutant.

  Finally, in [mSA] / [mAP] = 2, in addition to the elution times of 9.7 mL and 11.1 mL, a new peak was observed at 8.2 mL. The estimated molecular weight was 691 kDa and was expected to be an (AP-SA) 4-AP aggregate. From the above results, the formation of a wire-like aggregate was suggested in view of the estimated molecular weight.

  Next, the results for biotin-PEO4-amine modified AP (219-221) -Q mutant are shown in FIG. 4 and Table 8.

  In addition to biotin-PEO4-amine modified AP (219-221) -Q mutant, [mSA] / [mAP] = 1/4, 1/2 and 1 are added, and streptavidin-biotin-PEO4- An aggregate of amine modified AP (219-221) -Q mutants was formed. As a result, an increase in particle diameter was observed with the addition of streptavidin.

  However, the particle diameter of the aggregate is maximum at 34.9 nm when [mSA] / [mAP] = 1/2, and 26.0 nm under the condition of [mSA] / [mAP] = 1 by further adding streptavidin. And the particle size has decreased.

  In order to conduct a deeper study on the particle size reduction behavior due to the addition of streptavidin, the sample after DLS measurement was run on SEC, and the ratio of streptavidin to biotin-PEO4-amine modified AP (219-221) -Q mutant The formation behavior of the aggregates was traced.

  From the obtained chart, peaks of biotin-PEO4-amine modified AP (219-221) -Q mutant and streptavidin were observed at elution volumes of 14.2 mL and 16.2 mL, respectively. As streptavidin was added to biotin-PEO4-amine modified AP (219-221) -Q mutant, the peak of biotin-PEO4-amine modified AP (219-221) -Q mutant decreased, and more Fast elution time = formation of aggregates with large molecular weight was confirmed. When [mSA] / [mAP] = 1/2, large aggregates were observed in which almost all of the aggregates were eluted in the excluded volume. Under the conditions of [mSA] / [mAP] = 1, The molecular weight per aggregate decreased to complement the DLS measurement results.

  Streptavidin and biotin-PEO4-amine modified AP (219-221) -Q variant due to the behavior of increasing, decreasing and increasing the particle size in the order of [mSA] / [mAP] = 1/4 to 1/2 It was considered that the formation of aggregates using slag does not grow endlessly by the addition of streptavidin, but that there exists a certain stabilized state (particles with a stable diameter and molecular weight). In combination with the results for the AP (219-221) -K2 mutant, it was speculated that steric hindrance due to the linker length of the biotinylated substrate greatly affected the particle size.

Experimental Example 6: Functional analysis of complex of various alkaline phosphatase mutants Biotin-QG modified AP (219-221) -K2 mutant and biotin-PEO4-amine modified AP (219-221) prepared in Experimental Example 5 above Functional analysis of the complex of -Q mutant and streptavidin was performed.

  Functional analysis was performed on the complexes obtained by setting the mixed amounts of both mutants and streptavidin as described above [mSA] / [mAP] = 1/4, 1/2, and 1, respectively. As controls, only biotin-QG modified AP (219-221) -K2 mutant and biotin-PEO4-amine modified AP (219-221) -Q mutant, which do not contain streptavidin, were used.

  As a further comparative experiment, a chemically modified alkaline phosphatase obtained by chemically modifying biotin with respect to alkaline phosphatase was used. A specific manufacturing method is as follows.

  Biotin- (AC) 2-OSu was prepared to 500 or 50 μM (100 mM borate buffer pH 9.0) for 5 μM alkaline phosphatase, and chemically modified biotinylation reaction at 25 ° C. for 5 hours. Went. The sample after the reaction was subjected to centrifugal filtration and addition of TBS buffer using a 10 kDa filter to remove unreacted reagents and complete the reaction.

  Next, a plate for ELISA measurement was prepared. Ovalbumin (OVA), an antigen prepared to be 0, 0.001, 0.01, 0.1, 1, 10, 100, 1000 μg / ml, is added to each well so as to be 100 μl / well, Solid-phase formation was performed by leaving still at 4 degreeC for 12 hours. Next, after washing 5 times with TBST, 200 μl / well of 2% BSA / TBS was added and blocking was performed by allowing to stand at 37 ° C. for 2 hours. After washing, a 10000-fold diluted solution of the primary antibody anti-OVA mouse antibody, 100 μl / well, was added and allowed to stand at 37 ° C. for 2 hours. After washing, a 10000-fold diluted solution of biotinylated anti-mouse IgG rabbit antibody as a secondary antibody was added at 100 μl / well and allowed to stand at 37 ° C. for 2 hours. After washing, the above-mentioned various alkaline phosphatase aggregates and chemically modified alkaline phosphatase solution were added at 100 μl / well and allowed to stand at 37 ° C. for 2 hours.

  Finally, after the prepared plate was washed, the activity was measured using p-nitrophenyl phosphate (p-NPP) as a substrate. The activity was measured under the conditions of 1 M Tris-HCl (pH 8) containing 5 mM p-NPP at 37 ° C., and the activity was measured according to the above-described method for tracking the absorption at 410 nm. In this experiment, the absorbance under the conditions without streptavidin is set to Noise, and the S / N ratio is calculated from each absorbance. The results are shown in FIG.

  First, in the case of using two biotin-modified alkaline phosphatase mutants, no signal was observed at [mSA] / [mAP] = 1/4, but these were in solution that formed an alkaline phosphatase aggregate. This is because the sample has an excessive amount of biotin groups. Since all the biotin binding sites of streptavidin were occupied, it was predicted that they could not bind to the biotinylated antibody.

  However, when [mSA] / [mAP] = 1/2, a signal is observed in either case, and the sample condition is that the biotin group is bound in spite of the excessive biotin group as before. Here, when the S / N ratio of [SA] / [AP] = 1/4 is seen again, it does not reach 2, but the signal intensity is certainly compared with [mSA] / [mAP] = 0 μg / ml. I could see the enhancement.

  Therefore, it was speculated that the alkaline phosphatase aggregate may bind to the anti-mouse IgG antibody by an equilibrium reaction between avidin and biotin. The weak signal of [mSA] / [mAP] = 1/4 suggests that the amount of aggregate that can interact with biotin present in the system is small.

  On the other hand, when [mSA] / [mAP] = 1, a strong signal was observed, and it was confirmed that the AP assembly bound to the biotinylated antibody. The above results are considered to correlate with the growth of aggregates by changing the ratio of [mSA] / [mAP] shown in the above table. That is, it was suggested that the formation of larger aggregates contributed to the increase in signal intensity in ELISA.

In addition, the signal of [mSA] / [mAP] = 2 is weak. This is probably due to the increase in the binding of free streptavidin due to the excessive presence of streptavidin in the system. If there is a certain optimal value for the particle size and the hypothesis that adding more streptavidin will reduce the particle size per aggregate is correct, the excess of streptavidin will bind to each OVA. It was thought that this was also caused by a decrease in the amount of alkaline phosphatase.

  This is also due to the difference in signal strength between [mSA] / [mAP] = 1/2, 1 between AP (219-221) -K (b) and AP (219-221) -Q (b). It was thought that it became.

  For the chemically modified alkaline phosphatase, only a weak signal increase was observed in all cases. This is presumably because the biotinylation rate increased due to chemical modification, and an excessive amount of biotin groups were present in the system as compared with enzyme-modified biotinylated alkaline phosphatase. In addition, there was a concern that the activity of alkaline phosphatase itself was reduced by chemical modification.

Experimental Example 7: Complex formation ability of various alkaline phosphatase mutants Experiments were conducted on the ability of the biotin-modified alkaline phosphatase produced in Experimental Example 4 to form an aggregate. Specifically, AP (219-221) -K2 mutant was biotinylated using biotin-QG, biotin-GGG-GGLQG and bis (biotin-GGG) -KGLQG, and further using MTG. is there.

  These biotinylated alkaline phosphatases were added on a streptavidin-coated plate to attempt to immobilize these biotinylated alkaline phosphatases on the plate.

  For immobilization, 100 μL of each biotinylated alkaline phosphatase prepared to 1 μM was added to a plate coated with streptavidin, left to stand at 4 ° C. for 30 minutes, and then washed with 200 μl of TBS buffer. Next, alkaline phosphatase activity was measured by the method described above. The result is shown in FIG.

  In FIG. 7, (1) using biotin-QG as a biotinylated substrate, (2) using biotin-GGG-GGLQG, and (3) using bis (biotin-GGG) -KGLQG. ). In addition, WT in FIG. 7 is obtained by using wild-type alkaline phosphatase in place of the AP (219-221) -K2 mutant.

  As described above, it was revealed that the newly biotinylated substrate synthesized this time was recognized by MTG, and that the ability to bind to streptavidin was maintained even when alkaline phosphatase was biotinylated.

  Therefore, it became clear that alkaline phosphatase aggregates were formed on the plate.

Experimental Example 8: Complex formation ability of EGFP variant The EGFP variant shown in SEQ ID NO: 13 having the His tag of the amino acid sequence shown in SEQ ID NO: 15 (MHHHHHH) at the N-terminus was prepared. The specific production method was performed in the same manner as the above-mentioned alkaline phosphatase mutant. The EGFP variant shown in SEQ ID NO: 13 contains the amino acid sequence shown in SEQ ID NO: 4 at its C-terminus.

  Subsequently, bis (biotin-GGG) -KGLQG was used as a biotin compound, and the EGFP mutant was biotinylated. As biotinylation conditions, 10 μM EGFP mutant, 200 μM biotin compound, and 0.5 U / mL MTG were mixed in TBS buffer and reacted at 25 ° C. for 1 hour. After the reaction, a biotinylated EGFP mutant was purified using a gel filtration column (G-25).

  The obtained biotinylated EGFP was added to a plate coated with streptavidin, and immobilization of these biotinylated alkaline phosphatases on the plate was attempted.

  The immobilization was performed in the same manner as the immobilization of alkaline phosphatase of Example 7 on a plate coated with streptavidin. After immobilization, the plate was observed using a fluorescence imaging apparatus. The result is shown in FIG.

  FIG. 8A is an imaging image, and FIG. 8B is a graph in which the fluorescence intensity obtained from the imaging image is calculated. In the figure, K tag EGFP indicates a mutant, and WT uses a wild type in place of the EGFP mutant.

  From the results shown in FIG. 8, it was revealed that the novel biotinylated substrate synthesized this time was recognized by MTG and biotinylated on the EGFP mutant, similarly to the alkaline phosphatase mutant. It was revealed that EGFP maintains the ability to bind to streptavidin even in a biotinylated state.

  Therefore, it was also revealed that EGFP aggregates were formed on the plate.

Claims (26)

Following formula (1)

(Where
n is 1 or 2,
X is O or NH, and when n is 2, X may be the same or different;
R ¹ is a direct bond or an amino acid residue, and the amino acid residue of R ¹ when n is 2 may be the same or different. R ² is a direct bond when n is 1. The amino acid residue of R ² in the case where n is 2 has an amino residue obtained by removing one hydrogen atom from the terminal amino group of the side chain, and the amino residue and R ¹ Is an amino acid residue having a peptide bond,
R ³ is a direct bond or an amino acid residue;
Each amino acid residue of R ¹ or R ³ is one amino acid residue, or an amino acid residue in which two or more amino acid residues are peptide-bonded. )
A biotin compound represented by It said amino acid residues of R ¹ or R ³ comprises at least one selected from the group consisting of glycine and leucine residue, a biotin compound as claimed in claim 1. The biotin compound according to claim 1 or 2, wherein the amino acid residue of R ² is a lysine residue. The R ¹ amino acid residue is an amino acid residue in which 6 or less amino acid residues are peptide-bonded, or the R ³ amino acid residue is an amino acid residue in which 6 or less amino acid residues are peptide-bonded The biotin compound according to any one of claims 1 to 3, which is a group. Following formula (2)

(Where
n is 1 or 2,
X is O or NH, and when n is 2, X may be the same or different;
R ¹ is a direct bond or an amino acid residue, and when n is 2, the amino acid residue of R ¹ may be the same or different. R ² is when n is 1, In the case where n is 2 and the amino acid residue of R ² is a direct bond, the amino acid residue of R ² has an amino residue obtained by removing one hydrogen atom from the terminal amino group of the side chain, and the amino residue and R ¹ carboxylic acid residue is an amino acid residue having a peptide bond,
R ⁴ is a direct bond or an amino acid residue;
Each amino acid residue of R ¹ or R ⁴ is one amino acid residue, or an amino acid residue in which two or more amino acid residues are peptide-bonded. The biotin compound according to any one of claims 1 to 4, which is represented by: The biotin compound according to claim 5, wherein the amino acid residue of R ¹ or R ⁴ includes at least one glycine residue. It said amino acid residue of R ² is, biotin compound as claimed in claim 5 or 6 lysine residues. The amino acid residue of R ^{1 is} an amino acid residue in which 6 or less amino acid residues are peptide-bonded, or the amino acid residue of R ^{4 is} an amino acid residue in which 5 or less amino acid residues are peptide-bonded The biotin compound according to any one of claims 5 to 7, which is a group. Following formula (3)

(Where
X is the same or different and is O or NH;
R ⁵ _, R ⁶ , and R ⁷ are the same or different and each is a direct bond or an amino acid residue;
The amino acid residues of R ⁵ _, R ⁶ , and R ⁷ are each an amino acid residue in which one or two or more amino acid residues are peptide-bonded. )
The biotin compound of any one of Claims 1-8 represented by these. The biotin compound according to claim 9, wherein the amino acid residue of R ⁵ , R ⁶ , or R ⁷ contains at least one glycine residue. The R ⁵ amino acid residue is an amino acid residue in which 6 or less amino acid residues are peptide-bonded, and the R ⁶ amino acid residue is an amino acid residue in which 6 or less amino acid residues are peptide-bonded The biotin compound according to claim 9 or 10, wherein the amino acid residue of R ^{7 is} an amino acid residue in which 5 or less amino acid residues are peptide-bonded. Following formula (4)

The biotin compound of Claim 1 represented by these. Following formula (5)

The biotin compound of Claim 5 represented by these. Following formula (6)

The biotin compound of Claim 9 represented by these. The biotin labeling agent with respect to protein or a peptide containing the biotin compound of any one of Claims 1-14. The biotin labeling agent according to claim 15, wherein the protein or peptide comprises one or more amino acid sequences represented by any of SEQ ID NOs: 1 to 4. A biotin labeling kit for a protein or peptide comprising the biotin labeling agent according to claim 15 or 16 and transglutaminase. The biotin labeling kit according to claim 17, wherein the transglutaminase is a microorganism-derived transglutaminase. A protein or peptide biotin labeling method comprising the following steps 1 to 3;
(1) Step 1 of mixing the biotin compound according to any one of claims 1 to 14 with the protein or peptide in the presence of transglutaminase,
(2) Step 2 where the mixture of Step 1 is subjected to transglutaminase optimal activity conditions,
(3) Step 3 of recovering biotin-labeled protein or biotin-labeled peptide from the mixture after Step 2. The method according to claim 19, wherein the protein or peptide comprises one or more amino acid sequences represented by any of SEQ ID NOs: 1 to 4. The method according to claim 19 or 20, wherein the transglutaminase is a microorganism-derived transglutaminase. A protein or peptide aggregate into which one or more of the amino acid sequences shown in any of SEQ ID NOs: 1 to 4 are inserted or substituted. The protein or peptide assembly according to claim 22, comprising the amino acid sequence represented by any of SEQ ID NOs: 8 to 13. The following method for producing a protein or peptide aggregate, comprising steps 1 to 3;
(1) Step 1 of mixing the protein or peptide variant according to claim 22 or 23, the biotin compound according to any one of claims 1 to 14, the avidin compound, and transglutaminase,
(2) Step 2 where the mixture of Step 1 is subjected to the optimal activity condition of the transglutaminase,
(3) Step 3 of recovering a protein or peptide aggregate from the mixture after Step 2. The method according to claim 24, wherein the transglutaminase is a microorganism-derived transglutaminase. A protein or peptide aggregate obtained by the method according to claim 24 or 25.

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