WO2013084526A1 - Composé de biotine, agent de marquage de biotine et agrégat de protéine - Google Patents

Composé de biotine, agent de marquage de biotine et agrégat de protéine Download PDF

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WO2013084526A1
WO2013084526A1 PCT/JP2012/065407 JP2012065407W WO2013084526A1 WO 2013084526 A1 WO2013084526 A1 WO 2013084526A1 JP 2012065407 W JP2012065407 W JP 2012065407W WO 2013084526 A1 WO2013084526 A1 WO 2013084526A1
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amino acid
acid residue
peptide
biotin
residue
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PCT/JP2012/065407
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English (en)
Japanese (ja)
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典穂 神谷
裕太郎 森
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国立大学法人九州大学
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Priority to JP2013548108A priority Critical patent/JP6226750B2/ja
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/185—Heterocyclic compounds containing sulfur atoms as ring hetero atoms in the condensed system
    • C12P17/186—Heterocyclic compounds containing sulfur atoms as ring hetero atoms in the condensed system containing a 2-oxo-thieno[3,4-d]imidazol nucleus, e.g. Biotin

Definitions

  • the present invention relates to a biotinylated compound, a biotin labeling agent, and a protein assembly.
  • Non-patent Document 1 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 2 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 Protein self-assembly plays an important role in nature, as exemplified by the formation of viruses, microtubules, flagella, actin fibers, and the like.
  • 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.
  • the first problem in self-assembly of proteins is selection of driving force for assembly.
  • non-covalent bonds such as ⁇ - ⁇ stacking, hydrogen bonds, and van der Waals forces are used. Due to their versatility and applicability, a receptor and a ligand for it can be used. Emphasis is placed on the self-assembly design used.
  • 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.
  • 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.
  • R 1 is a direct bond or an amino acid residue
  • 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.
  • 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 The biotin compound according to Item 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 Item 4.
  • 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.
  • Item 5 The biotin compound according to any one of Items 1 to 4, which is represented by:
  • 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 The biotin compound according to Item 5 or 6, wherein the amino acid residue of R 2 is a lysine residue.
  • 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.
  • 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.
  • 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.
  • the R 5 amino acid residue is an amino acid residue in which 6 or less amino acid residues are peptide-bonded
  • the R 6 amino acid residue is an amino acid residue in which 6 or less amino acid residues are peptide-bonded Item 11.
  • 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 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.
  • Item 19 A biotin labeling method of protein or peptide 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 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 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 The protein or peptide variant according to Item 22, comprising the amino acid sequence shown in any one of SEQ ID NOs: 8 to 13.
  • Item 24 The following method for producing a protein or peptide assembly, comprising steps 1 to 3; (1) Step 1 of mixing the protein or peptide variant shown in 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 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.
  • Biotin Compound The biotin compound of the present invention is represented by the formula (1).
  • n 1 or 2.
  • X is O or NH. Further, when n is 2, X may be the same or different.
  • R 1 is a direct bond or an amino acid residue.
  • n 2
  • the amino acid residues of R 1 may be the same or different.
  • R 3 is a direct bond or an amino acid residue.
  • 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.
  • 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.)
  • 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.
  • amino acids include 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.
  • 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.
  • amino and carboxy residues are also referred to herein as amino and carboxy residues, respectively.
  • 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.
  • R 2 when n is 1, R 2 is a direct bond.
  • 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.
  • 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.
  • R 1 is a direct bond
  • 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.
  • 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.
  • 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. is doing.
  • 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 .
  • 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 .
  • R 2 is peptide-bonded to R 1 at two positions.
  • the R 2 when n is 2 is not particularly limited, but is preferably a lysine residue.
  • 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. .
  • 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.
  • 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.
  • biotin compound represented by 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).
  • biotin-QG the biotin compound represented by the formula (4) may be referred to as biotin-QG.
  • 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.
  • n 1 or 2.
  • X is O or NH. Further, when n is 2, X may be the same or different.
  • R 1 is a direct bond or an amino acid residue.
  • n 2
  • the amino acid residues of R 1 may be the same or different.
  • R 4 is a direct bond or an amino acid residue.
  • 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.
  • amino acid residue in formula (2) and the amino acid residue in which two or more amino acids are peptide-bonded are the same as those in formula (1).
  • 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.
  • R 2 when n is 1, R 2 is a direct bond.
  • 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.
  • R 4 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.
  • R 1 is a direct bond
  • 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.
  • 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.
  • 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. is doing.
  • 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 .
  • 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 .
  • R 2 is peptide-bonded to R 1 at two positions.
  • R 2 when n is 2 is not particularly limited, but is preferably a lysine residue.
  • 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.
  • 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.
  • 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.
  • biotin compound represented by formula (5) 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.
  • the biotin compound represented by the formula (5) may be referred to as biotin-GGG-GGLQG.
  • biotin compound represented by the formula (1) is a biotin compound represented by the formula (3).
  • n 2
  • R 1 corresponds to R 5 and R 6 respectively
  • R 2 is a lysine residue
  • 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.
  • amino acid residue in which one hydrogen atom from the amino group of ⁇ -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).
  • 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).
  • X is O or NH, and these may be the same or different.
  • R 5 , R 6 , or R 7 is a direct bond or an amino acid residue.
  • 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 and the amino acid residue in which two or more amino acids are peptide-bonded are the same as those in the formula (1) or the formula (2).
  • R 5 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 ⁇ -terminal amino group of the side chain of the lysine residue.
  • R 6 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the most preferred biotin compound is a biotin compound represented by formula (6).
  • 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.
  • 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 a solid phase synthesis method.
  • a specific peptide synthesis method includes a method according to the following procedure.
  • 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 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.
  • biotin compounds such as N-BiotinyL-3, 6, 9-TRIOXAUNDECANENE-1, 11-DIAMINE, biotin cadaverine and the like are also useful as the biotin labeling agent of the present invention. These can be obtained from Molecular Biosciences.
  • the biotin labeling agent of the present invention may contain, in addition to the above-described biotin compound of the present invention, a known substance that is usually contained in a reagent as long as biotinylation to an amino acid or protein is not hindered.
  • the above biotin compound may be contained in an amount of 0.001 to 99.9 parts by weight.
  • the protein or peptide that is the target of the biotin labeling agent of the present invention is not particularly limited, but preferably includes any one or more of the amino acid sequences shown in SEQ ID NOs: 1 to 4, for example.
  • 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
  • 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.
  • part between and is included is mentioned.
  • the protein when it is EGFP, it may be inserted into the C-terminus and included.
  • 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.
  • 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 containing 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.
  • 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.
  • Insertion may be inserted at the N-terminal, C-terminal, or other site of the protein or peptide.
  • Substitution may be substituted with the amino acid sequence at the N-terminal, C-terminal, or other site of the protein or peptide
  • 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.
  • part between residues is mentioned.
  • 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.
  • 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.
  • the identity of amino acid sequences 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.
  • 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 later 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
  • the biotin labeling method of the present invention is a biotin labeling method of a protein or peptide, that is, a method of biotinylating a protein or peptide, and includes the following steps 1 to 3.
  • 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 where the mixture of Step 1 is placed under optimal activity conditions for transglutaminase.
  • Step 3 of recovering biotin-labeled protein or biotin-labeled peptide from the mixture after Step 2.
  • 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 a suitable solvent, and the kind thereof is not particularly limited.
  • a well-known buffer frequently used in biochemical experiments may be used.
  • 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.
  • 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.
  • biotin compound and the protein or peptide may be mixed in an amount that is usually about 1: 1 to 1:20 in molar ratio.
  • 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.
  • the condition in Step 2 is a condition under which such a reaction proceeds successfully.
  • 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 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.
  • 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.
  • Step 2 wherein the mixture of Step 1 is subjected to the optimal activity condition of the transglutaminase.
  • Step 3 for recovering the alkaline phosphatase aggregate after Step 3 above.
  • 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 described above.
  • 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.
  • 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.
  • transglutaminase when a protein or peptide, a biotin compound, and transglutaminase are mixed in advance, it is preferable that the mixture is placed under conditions for optimal activity of transglutaminase after mixing. Specific conditions may be the same as those described in step 2.
  • step 1 includes the formation of the complex
  • biotin compound has two biotinyl groups
  • 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.
  • step 1 the protein or peptide, biotin compound, avidin compound, and transglutaminase may be mixed in an appropriate solvent.
  • a specific solvent is not particularly limited, a known buffer used in biochemical experiments may be used.
  • 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.
  • 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.
  • 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 is a step of recovering the protein or peptide aggregate after Step 2.
  • 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.
  • the method may be the same as the above-described method for producing a protein or peptide variant according to the present invention.
  • 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.
  • biotin compound of the present invention is listed below. Needless to say, the biotin compound of the present invention is not limited to those having all the following effects.
  • biotin compound of the present invention can be specifically bound 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 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.
  • 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 of various alkaline phosphatase mutants modified with FITC, stained with CBB after SDS-PAGE.
  • C Fluorescent photographic images after SDS-PAGE of various alkaline phosphatase mutants modified with FITC.
  • 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 of various alkaline phosphatase mutants modified with FITC, stained with CBB after SDS-PAGE.
  • Example 1 Preparation of alkaline phosphatase mutant
  • the alkaline phosphatase mutant shown in Table 3 below was prepared. Specifically, nucleotide sequences 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 are used as vectors for expression of E. coli (pET22b ( An alkaline phosphatase mutant expression vector incorporated in (+)) was prepared. E. coli BL21 (DE3) strain was transformed with such a vector.
  • pET22b An alkaline phosphatase mutant expression vector incorporated in (+)
  • This transformant was collected after culturing in an appropriate medium, 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. A Ni-NTA column was used in the purification process.
  • 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.
  • p-NPP p-nitrophenyl phosphate
  • the activity was measured at 25 ° C., and 10 ⁇ l of various alkaline phosphatase solutions (concentration 2 ⁇ M) were added to 990 ⁇ l 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.
  • 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- ⁇ -Ala-QG (see Org. Biomol. Chem., 2009, 7, 3407-3412). This is a compound in which FITC is covalently bonded to the N-terminus of the Ala-Gln-Gly tripeptide, and is known as a substrate for transglutaminase.
  • alkaline phosphatase mutant AP (219-221) -Q prepared 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.
  • 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.
  • the RK sequence was more suitable for the sequence around the lysine residue in MTG recognition.
  • the insertion site it is considered that the 219th to 221nd positions are lower in steric hindrance than the 91th to 93rd positions, and the MTG is easy to approach.
  • 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.
  • AP (219-221) -K2 biotinylated substrate, and MTG were added to TBS buffer so that the final concentrations were 0.5 mg / ml (10 ⁇ M), 200 ⁇ M, and 0.5 U / ml. was stirred well and allowed to stand at 25 ° C.
  • 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 100 ml of 1N NaOH was added to each and stirred vigorously. 20 ml of egg white-derived avidin solution (10 mg) was placed in a volumetric flask, then 600 ml of HABA solution was added, and the volume was adjusted with PBS to prepare a HABA-Avidin solution.
  • HABA 4-hydroxy-azobenzene-2'-carboxylic acid
  • biotinylated alkaline phosphatase was measured by two methods.
  • biotinylation 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.
  • 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.
  • alkaline phosphatase activity was observed on the plate, so the ability of biotin modified with alkaline phosphatase to bind to streptavidin was confirmed.
  • alkaline phosphatase mutants were active even when bound to streptavidin. It was confirmed that it was maintained.
  • Biotinylation of AP (219-221) -Q with MTG was performed using biotin- (PEO) 4-amine as a biotinylated substrate.
  • the enzyme reaction was carried out in TBS buffer with AP (219-221) -Q, biotin- (PEO) 4 ⁇ so that the final concentrations were 0.5 mg / ml (10 ⁇ M), 200 ⁇ M, and 0.5 U / ml, respectively. Amine and MTG were added, and they were stirred well and allowed to stand at 25 ° C.
  • the biotinylation rate of AP (219-221) -Q was 0.97 ⁇ 0.04, and it can be said that almost quantitative biotinylation was achieved.
  • the results of attempts to immobilize AP (219-221) -Q on the streptavidin plate revealed that the activity of alkaline phosphatase was maintained without any problem on the plate. .
  • 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
  • concentration was 4 ⁇ M.
  • the solution after DLS measurement was fractionated by size exclusion column chromatography (SEC), and the behavior of formation of the formed aggregate 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.
  • 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.
  • 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 of streptavidin. On the other hand, biotin-QG modified AP (219-221) -K2 mutant seems to be bound respectively.
  • biotin-GGG-GGLQG-modified AP (219-221) -K2 mutant is a linear biotinylated substrate like 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.
  • the particle diameter of the aggregate was decreased by increasing the linker length.
  • 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 The functional analysis of the complex of -Q mutant and streptavidin was performed.
  • Biotin- (AC) 2-OSu is adjusted to 500 or 50 ⁇ M (100 mM borate buffer, pH 9.0) against 5 ⁇ M 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.
  • Ovalbumin an antigen prepared to be 0, 0.001, 0.01, 0.1, 1, 10, 100, 1000 ⁇ g / ml, is added to each well so as to be 100 ⁇ l / well, Solid-phase formation was performed by leaving still at 4 degreeC for 12 hours.
  • 200 ⁇ l / well of 2% BSA / TBS was added and blocking was performed by allowing to stand at 37 ° C. for 2 hours.
  • a 10000-fold diluted solution of anti-OVA mouse antibody which is the primary antibody, was added at 100 ⁇ l / well, and left at 37 ° C. for 2 hours.
  • 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.
  • the absorbance under conditions without streptavidin is set to Noise, and the S / N ratio is calculated from each absorbance. The results are shown in FIG.
  • 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.
  • Experimental Example 7 Complex formation ability of various alkaline phosphatase mutants
  • AP (219-221) -K2 mutant was biotinylated using biotin-QG, biotin-GGG-GGLQG and bis (biotin-GGG) -KGLQG, and further using MTG. is there.
  • biotinylated alkaline phosphatases were added onto a plate coated with streptavidin, and an attempt was made to immobilize these biotinylated alkaline phosphatases on the plate.
  • the biotinylated substrate using biotin-QG is (1), the biotin-GGG-GGLQG is used (2), and the bis (biotin-GGG) -KGLQG is used (3). ).
  • WT in FIG. 7 is obtained by using wild-type alkaline phosphatase in place of the AP (219-221) -K2 mutant.
  • Experimental Example 8 Complex formation ability of EGFP variant
  • 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.
  • biotin-GGG bis (biotin-GGG) -KGLQG was used as a biotin compound, and the EGFP mutant was biotinylated.
  • biotinylation conditions 10 ⁇ M EGFP mutant, 200 ⁇ M biotin compound and 0.5 U / mL MTG were mixed in TBS buffer and reacted at 25 ° C. for 1 hour. After the reaction, the biotinylated EGFP mutant was purified using a gel filtration column (G-25).
  • biotinylated EGFP was added to a plate coated with streptavidin, and an attempt was made to immobilize these biotinylated alkaline phosphatases on the plate.
  • the immobilization was carried out in the same manner as the immobilization of the alkaline phosphatase of Example 7 on the 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
  • FIG. 8B is a graph in which the fluorescence intensity obtained from the imaging image is calculated.
  • K tag EGFP indicates a mutant
  • WT uses a wild type in place of the EGFP mutant.

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Abstract

Le problème décrit par la présente invention est de proposer un agrégat de protéine qui a une fonction et au problème de fournir un composé de biotine qui est efficace pour la production de l'agrégat de protéine. La présente invention concerne un composé de biotine qui est représenté par la formule (1) comme solution aux problèmes mentionnés ci-dessus. (Dans la formule, n est 1 ou 2 ; X représente O ou NH, et dans des cas où n est 2, les groupes X peuvent être identiques entre eux ou différents les uns des autres ; R1 représente une liaison directe ou un reste d'acide aminé ; et dans des cas où n est 2, les restes d'acides aminés des groupes R1 peuvent être identiques entre eux ou différents les uns des autres ; dans des cas ou n est 1, R2 représente une liaison directe, alors que dans des cas où n est 2, les restes d'acides aminés des groupes R2 sont des restes d'acides aminés, dont chacun a un reste amino qui est obtenu par élimination d'un atome d'hydrogène à partir du groupe amino à l'extrémité d'une chaîne latérale et dans lequel le reste amino est lié au reste acide carboxylique du groupe R1 par une liaison peptidique ; R3 représente une liaison directe ou un reste d'acide aminé ; et le reste d'acide aminé du groupe R1 ou du groupe R3 est un reste d'acide aminé unique ou un reste d'acide aminé dans lequel au moins deux restes d'acides aminés sont liés ensemble par une liaison peptidique).
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JP2015229672A (ja) * 2014-06-06 2015-12-21 国立大学法人九州大学 ビオチン化合物、ビオチン標識化剤及びタンパク質集合体
WO2018151301A1 (fr) * 2017-02-17 2018-08-23 三井化学株式会社 Procédé d'identification d'une protéine cible de médicament potentielle utile pour le développement d'un médicament à base d'anticorps, et procédé de production d'anticorps dirigé contre la protéine cible
JPWO2018151301A1 (ja) * 2017-02-17 2019-12-12 三井化学株式会社 抗体医薬の開発に資する創薬標的タンパク質の同定方法及び標的タンパク質に対する抗体の製造方法
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JP7174951B2 (ja) 2017-02-17 2022-11-18 三井化学株式会社 抗体医薬の開発に資する創薬標的タンパク質の同定方法及び標的タンパク質に対する抗体の製造方法

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