JP2012116812A - Reagent for promoting stereostructure-formation of protein using disulfide bond exchange reaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a disulfide exchange reaction promoter for promoting stereostructure-formation reaction of protein.SOLUTION: The disulfide exchange reaction promoter comprises a Cys-Arg dipeptide derivative represented by a formula as reduction reagent and an Arg-Cys derivative represented by a formula as oxidation reagent. A method for producing protein comprises a refolding step of forming the stereostructure of protein by use of the disulfide exchange reaction promotor or the stereostructure-forming agent of protein.

Description

  The present invention relates to a disulfide exchange reaction accelerator for the purpose of promoting a three-dimensional structure formation reaction of a protein, a three-dimensional structure formation accelerator for a protein containing the disulfide exchange reaction accelerator, and the disulfide exchange reaction accelerator (or the The present invention relates to a method for producing a protein, comprising a step of forming a protein three-dimensional structure using a protein three-dimensional structure formation accelerator.

  Elucidation of the structure and function of related proteins is extremely important for research and development of disease diagnosis and treatment technologies, and drug discovery, and a large amount of proteins as therapeutic agents must be provided. In particular, since a protein can express its specific function as an enzyme or the like only after a three-dimensional structure is formed (folded), there is a synthesis / production method capable of quickly obtaining a target protein with high yield. It has been demanded.

  Currently, various proteins are synthesized and produced by various methods including genetic engineering techniques. However, it is usually not easy to obtain the correct three-dimensional structure of the target protein in a high yield, and the association between the polypeptide chains constituting the protein is the cause, particularly under high concentration conditions for the purpose of mass synthesis. Prone to protein aggregation and precipitation. For example, as a commonly used method for synthesizing a recombinant protein, a method for artificially expressing a target protein in a prokaryotic organism such as Escherichia coli that has been genetically manipulated or a eukaryotic organism such as yeast or a cell-free extraction system, the target protein is It is often obtained as a protein in an inactive state, that is, an inclusion body (inactive aggregate), which is inadvertently formed into a three-dimensional structure (misfolded). For this reason, after denatured unfolding, a method of refolding to the original three-dimensional structure is required.

  So far, reagents (agents) for promoting the three-dimensional structure formation (folding) reaction of proteins have been known (Patent Documents 1 and 2). However, in the case of a protein containing a disulfide bond in a molecule such as lysozyme. However, the disulfide bond is not correctly bonded, an incorrect three-dimensional structure is formed, and protein aggregation occurs, resulting in a problem that the yield of the target protein is low.

  Here, the correct disulfide bond formation and conversion of the protein to the natural conformation is the result of a reversible thiol (SH) / disulfide (SS) exchange reaction in the protein conformation (folding), and this Is thermodynamically and kinetically related to the redox potential in biological environments.

  Glutathione (γ-Glu-Cys-Gly) is one of the most abundant thiol compounds present in cells and plays a major role in the formation of disulfide bonds in proteins within the endoplasmic reticulum. GSSG (oxidized glutathione) functions as an oxidizing reagent in the formation of disulfide bonds in proteins, while GSH (reduced glutathione) functions as a reducing reagent that cleaves accidentally cross-linked disulfide bonds in proteins. These actions result in the formation of a thermodynamically stable three-dimensional structure of the protein (Non-patent Document 1). Therefore, glutathione is widely used in the three-dimensional structure (folding) reaction of disulfide-containing proteins at the laboratory level (Non-patent Documents 2 and 3).

  In general, a protein conformation (folding) reaction without disulfide bond formation is completed within a few minutes. However, a disulfide bond-containing protein requires several hours to form a three-dimensional structure. The main reason is that a disulfide bond is formed at the wrong position during the formation of the three-dimensional structure, and a long reaction time is required for the cleavage and the formation reaction of the natural disulfide bond (Non-Patent Document). 4). That is, in general, this is because the disulfide bond exchange reaction in this reaction intermediate is the rate-determining step for the correct three-dimensional structure formation. Therefore, the three-dimensional structure formation reaction of a protein having a disulfide bond usually causes a disulfide bond exchange reaction of a reaction intermediate having a wrong disulfide bond in the presence of a redox reagent, resulting in a natural three-dimensional structure. The transition to can be facilitated. At this time, the redox reagent performs a disulfide exchange reaction with the reaction intermediate to form a molecular species having a crossed disulfide bond with the intermediate. Therefore, in order to accelerate the formation of the correct natural disulfide bond, it is necessary to shorten the lifetime of the crossed disulfide intermediate, that is, to increase the reaction activity of the crossed disulfide bond. For this reason, it is necessary to develop a reagent that focuses on the chemical properties of the reaction intermediate, instead of developing a reaction reagent focusing on the properties (redox potential) of the redox reagent itself as in the prior art.

  There are two types of reaction reagents for purification of proteins having disulfide bonds. This is a reagent that protects the SH group of cysteine and increases the solubility of the denatured protein. For example, TAPS-sulfonate (Wako Pure Chemical Industries) and S-sulfonated reagent are known. However, in any reagent, after refining the protein chemically modified by them, a refolding reaction must be performed, and the operation is complicated. Moreover, it is necessary to separately add a reducing reagent (for example, 2-mercaptoethanol, reduced glutathione (GSH)) to form a disulfide bond after isolation, which is not a fundamental solution.

That is, the disulfide bond forming reaction requires a so-called redox reagent (for example, a glutathione (GSH / GSSG) or redox system of a thiol compound (DTT _red / DTT _ox , Cys _red / Cys _ox )). There are problems such as a low yield of the target protein having the correct steric structure or a slow disulfide bond exchange reaction (Non-patent Documents 5 and 6). In addition, regarding the properties of glutathione, only the oxidation-reduction potential is interpreted, and the role of individual amino acids contained or the reaction activity is not optimized.

JP 2009-073815 A JP 2010-132649 A

Hwang, C. et.al., Science, 257, 1496-1502 (1992) Konishi, Y. et.al., Biochemistry, 21, 4734-4740 (1982) Lyles, M. et.al., Biochemistry, 30, 613-619 (1991) Arolas, J. L., et. Al., Trends Biochem. Sci., 31, 292-301 (2006) Wedemeyer, W. J., et.al., Biochemistry, 39, 4207-4216 (2000) Chatrenet, B. et. Al., J. Biol. Chem., 268, 20998-20996 (1993)

  The present invention improves the solubility of the intermediate of the three-dimensional structure formation reaction, and promotes the disulfide exchange reaction by localizing a positive charge around the sulfur atom on the cross-disulfide intermediate that is the reaction center. An object of the present invention is to provide an agent (reagent) capable of shortening the existence time of a reaction intermediate having low solubility, suppressing aggregation of the intermediate, and efficiently and quickly forming a correct natural three-dimensional structure. .

The rate-limiting step of the protein folding (stereostructure formation) reaction using the redox reagent is a nucleophilic reaction of the thiol group in the protein (RS ⁻ ) to the crossed disulfide bond between the protein and the reagent. Therefore, it can be expected that the reaction rate of the nucleophilic reaction by the thiol group having a negative charge can be increased by localizing the positive charge around the sulfur atom of the crossed disulfide bond. The present inventor can localize a positive charge around a sulfur atom by arranging a molecule (group) having a positively charged functional group in the vicinity of a cysteine residue in the compound represented by the formula (I). Thought. And as a result of earnest research, it is a peptide having an arginine residue having a positively charged functional group adjacent to the cysteine residue or a derivative thereof, etc., which is neutral to alkaline corresponding to the condition of the three-dimensional structure formation reaction. the sum of the charge of the functional groups ± 0 or more in the whole molecule ^- a compound which is (thiol group S is not considered) is, functions as an excellent disulfide exchange reaction accelerator, as a result, a three-dimensional structure formation accelerator protein I found it to work. That is, the present invention is as follows.

［式中、
Ｘ_１およびＸ_２はそれぞれ独立して、水素原子、炭素数１〜６のアルキル基、炭素数１〜６のアシル基、炭素数１〜６のアルコキシカルボニル基、アラルキルオキシカルボニル基、天然型アミノ酸残基もしくは非天然型アミノ酸残基またはそれらの誘導体、天然型アミノ酸もしくは非天然型アミノ酸またはそれらの誘導体からなるペプチド残基またはそのペプチド残基誘導体であり；
Ｙは、ヒドロキシル基、ヒドラジノ基、炭素数１〜６のアルコキシ基、適宜１もしくは２個の置換基（当該置換基は、炭素数１〜６のアルキル基、炭素数１〜６のアシル基、炭素数１〜６のアルコキシカルボニル基、およびアラルキルオキシカルボニル基からなる群から独立して選ばれる）で置換されたアミノ基、天然型アミノ酸残基もしくは非天然型アミノ酸残基またはそれらの誘導体、天然型アミノ酸もしくは非天然型アミノ酸またはそれらの誘導体からなるペプチド残基またはそのペプチド残基誘導体であり；
Ａｒｇは、Ｃｙｓとα結合またはγ結合し；ここで、
中性または塩基性において分子全体における官能基の電荷の総和が０以上である］
で示されるペプチド、その立体異性体もしくはそのペプチド誘導体、またはそれらの塩もしくは溶媒和物から選択される少なくとも１種の化合物；および、適宜、酸化試薬としての式（II）：

［式中、
Ｘ_１、Ｘ_２およびＹが前記に定義する通りであり；
Ａｒｇが、Ｃｙｓとα結合またはγ結合し、ここで、
中性または塩基性において分子全体における官能基の電荷の総和が０以上である］
で示されるペプチド、その立体異性体もしくはそのペプチド誘導体、またはそれらの塩もしくは溶媒和物から選択される少なくとも１種の化合物、
を含む、ジスルフィド交換反応促進剤。 Disulfide exchange accelerator [1] Formula (I) as a reducing reagent:

[Where:
X ₁ and X ₂ are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an acyl group having 1 to 6 carbon atoms, an alkoxycarbonyl group having 1 to 6 carbon atoms, an aralkyloxycarbonyl group, a natural amino acid A residue or a non-natural amino acid residue or a derivative thereof, a peptide residue consisting of a natural amino acid or a non-natural amino acid or a derivative thereof, or a peptide residue derivative thereof;
Y is a hydroxyl group, a hydrazino group, an alkoxy group having 1 to 6 carbon atoms, an appropriate 1 or 2 substituent (the substituent is an alkyl group having 1 to 6 carbon atoms, an acyl group having 1 to 6 carbon atoms, An amino group substituted with an alkoxycarbonyl group having 1 to 6 carbon atoms and an aralkyloxycarbonyl group independently selected from natural amino acid residues or non-natural amino acid residues or derivatives thereof, natural A peptide residue or a peptide residue derivative thereof consisting of a type amino acid or a non-natural amino acid or a derivative thereof;
Arg is α- or γ-bonded to Cys;
The total charge of functional groups in the whole molecule is 0 or more in neutral or basic]
Or at least one compound selected from the stereoisomers or peptide derivatives thereof, or salts or solvates thereof; and, optionally, formula (II) as an oxidizing reagent:

[Where:
X ₁ , X ₂ and Y are as defined above;
Arg is α- or γ-bonded to Cys, where
The total charge of functional groups in the whole molecule is 0 or more in neutral or basic]
At least one compound selected from a peptide represented by the following, a stereoisomer or peptide derivative thereof, or a salt or solvate thereof:
A disulfide exchange reaction accelerator.

[2] X ₁ and X ₂ are each independently a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an acyl group having 1 to 4 carbon atoms, an alkoxycarbonyl group having 1 to 4 carbon atoms, neutral or basic Natural amino acid residues or derivatives thereof;
Y is a hydroxyl group, a hydrazino group, an alkoxy group having 1 to 4 carbon atoms, an appropriate 1 or 2 substituent (the substituent is an alkyl group having 1 to 4 carbon atoms, an acyl group having 1 to 4 carbon atoms, An amino group substituted with an alkoxycarbonyl group having 1 to 4 carbon atoms and an aralkyloxycarbonyl group independently selected from the group consisting of neutral or basic natural amino acid residues or derivatives thereof,
[1] The disulfide exchange reaction accelerator according to [1].

[3] The disulfide exchange reaction accelerator according to any one of [1] or [2], wherein the natural amino acid residue is a basic amino acid residue selected from the group consisting of L-Arg and L-Lys.

[4] The disulfide exchange accelerator according to any one of [1] to [3], wherein the total charge of functional groups in the whole molecule is 1 or more in neutral or basic.

[5] The disulfide exchange reaction accelerator according to any one of [1] to [4], wherein Arg is α-bonded to Cys.

[6] The disulfide exchange reaction accelerator according to any one of [1] to [5], further comprising at least one other oxidizing reagent.

[7] The disulfide exchange reaction accelerator according to any one of [1] to [6], wherein the weight ratio of the reducing reagent to the oxidizing reagent is 0.01 to 20.

[8] [1] to [1] further including an additive selected from the group consisting of a buffer solution, salts, buffer agents, bases, acids, organic solvents, surfactants, pH adjusters, and protein stabilizers as appropriate. [7] The disulfide exchange reaction accelerator according to any one of [7].

[9] (a) The reducing reagent and an additive selected from the group consisting of the buffer, salts, buffers, bases, acids, organic solvents, surfactants, pH adjusters, and protein stabilizers as appropriate; And
(B) the reducing reagent, and an additive selected from the group consisting of the buffer, salts, buffers, bases, acids, organic solvents, surfactants, pH adjusters, and protein stabilizers as appropriate;
The disulfide exchange reaction promoter according to any one of [1] to [8], wherein the disulfide exchange reaction promoter is contained in the same container form or in a separately packaged form.

Protein three-dimensional structure forming agent [10] A protein three-dimensional structure comprising the disulfide exchange reaction accelerator according to any one of [1] to [9] and, optionally, at least one further protein three-dimensional structure forming accelerator. Forming agent.

[11] The protein three-dimensional structure forming agent according to [10], wherein the three-dimensional structure formation of the protein is refolding.

Protein production method [12] [1] to [9] The disulfide exchange reaction promoter according to any one of [1] to [9] or the protein three-dimensional structure forming agent according to any one of [10] or [11] A method for producing a protein, comprising a step of forming a three-dimensional structure.

で示されるペプチド、その立体異性体もしくはそのペプチド誘導体、またはそれらの塩もしくは溶媒和物から選択される少なくとも１種。 The present invention further provides the following aspects of the invention.
New peptide [13] formula:

Or at least one selected from a stereoisomer or peptide derivative thereof, or a salt or solvate thereof.

[14] The peptide according to [13], which is a peptide composed of L-Arg and L-Cys.

  By using the disulfide exchange reaction accelerator of the present invention, the correct natural three-dimensional structure can be formed efficiently and rapidly, and the yield of the target natural protein can be greatly improved.

It is a figure which shows disulfide bond formation in the folding of a reduction | restoration modification prouroguaniline using a folding agent (2mM reduced glutathione / 1mM oxidized glutathione). It is drawing which shows disulfide bond formation in the folding of reduction | denaturation modification | denaturation prouroguanylin using a folding agent (2mM reduction type | mold ECR / 1mM reduction type | mold ECR). It is drawing (experimental example 2) which shows disulfide bond formation in the folding of reduction | denaturation modification prouroguanylin using a folding agent (2mM reduction type | mold RCG / 1mM reduction type | mold RCG). The results of comparing the effects on the secondary structure formation by circular dichroism spectrum analysis in folding using folding agents (2 mM reduced glutathione / 1 mM oxidized glutathione, 2 mM reduced RCG / 1 mM reduced RCG) are shown ( Example 3) Drawing. Various concentrations using folding agents (open bar; 2mM reduced glutathione / 1mM oxidized glutathione, cross-hatched bar; 2mM reduced ECR / 1mM reduced ECR, shaded bar; 2mM reduced RCG / 1mM reduced RCG) It is drawing (experimental example 4) which shows the result of having compared the folding yield in the reduction | denaturation modification | denaturation lysozyme of (0.1-1.6 mg / mL). Using a folding agent (open bar; 2 mM reduced glutathione / 1 mM oxidized glutathione, shaded bar; 2 mM reduced RCG / 1 mM reduced RCG) in various concentrations (0.1 to 0.4 mg / mL) of reduced modified prouroguanylin It is drawing (experimental example 5) which shows the result of having compared the folding yield. It is a figure which shows the comparison (experimental example 6) of the selective ability of the correct disulfide bond at the time of folding in a reduction | denaturation modification | denaturation prourologaniline using a folding agent (1 mM oxidized glutathione, 1 mM oxidized RCG).

The present invention is described in further detail below.
(Definition)
The definitions of terms used in the present specification and claims are shown below. Unless otherwise indicated, the first definition given for a group or term herein applies to the group or term herein individually or as part of another group.

で示されるペプチド、すなわち式（Ｉ−Ａ）および（Ｉ−Ｂ）で示されるペプチドを意味する。ここで、上記一般式の表示は、当該有機化学分野で通常使用されるとおり、各アミノ酸残基のアミノ基を左側に、カルボキシル基を右側に配置する。また、「Ｃｙｓ」および「Ａｒｇ」はそれぞれシステイン残基およびアルギニン残基を意味する。よって、式（Ｉ−Ａ）のペプチドは、システインのカルボキシル基とアルギニンのアミノ基とがペプチド結合したジペプチドをコア部分として有する化合物を、一方で式（Ｉ−Ｂ）のペプチドはアルギニンのカルボキシル基とシステインのアミノ基とがペプチド結合したジペプチドをコア部分として有する化合物を意味する。
また、当該式（Ｉ）で示されるペプチドは、システイン残基の側鎖にあるチオール基が遊離であることから、本明細書中「還元型ペプチド」とも呼称し、ジスルフィド交換反応における還元試薬として作用する。 I. Disulfide exchange method
The reducing reagent term “peptide represented by the formula (I)” means the following formula:

In other words, peptides represented by the formulas (IA) and (IB). Here, the display of the above general formula is that the amino group of each amino acid residue is arranged on the left side and the carboxyl group is arranged on the right side as normally used in the field of organic chemistry. “Cys” and “Arg” mean a cysteine residue and an arginine residue, respectively. Thus, the peptide of formula (IA) is a compound having as its core part a dipeptide in which the carboxyl group of cysteine and the amino group of arginine are peptide-bound, while the peptide of formula (IB) is a carboxyl group of arginine. And the amino group of cysteine is a compound having a peptide-bonded dipeptide as a core part.
Moreover, since the thiol group in the side chain of the cysteine residue is free, the peptide represented by the formula (I) is also referred to as “reduced peptide” in the present specification, and is used as a reducing reagent in the disulfide exchange reaction. Works.

ここで、本発明におけるジスルフィド交換反応においては、式（Ｉ）における構成アミノ酸の立体配置は限定されるものではない。よって、構成アミノ酸であるシステインおよびアルギニン、並びにＸ、ＹおよびＺ基がとり得る天然アミノ酸などはＬ−体もしくはＤ−体またはＤＬ−ラセミ体のいずれであってもよい。よって、分子全体でこれらアミノ酸の各立体異性体のいずれかの組み合わせを含み、用語「立体異性体」とはこれらの組み合わせを含むことを意図する。 In the above formula (I), Arg can be formed by Cys with either an α bond or a γ bond. However, from the viewpoint of ease of production (especially chemical synthesis) and availability of raw material, α Bonding is preferred. Here, the case where an α bond or a γ bond is formed with Cys refers to a case where the amino group part of the main chain or the side chain of arginine is bonded to the carboxyl group part of Cys. A specific structural formula is shown below.

Here, in the disulfide exchange reaction in the present invention, the configuration of the constituent amino acids in the formula (I) is not limited. Therefore, cysteine and arginine, which are constituent amino acids, and natural amino acids that can be taken by the X, Y, and Z groups may be either L-form, D-form, or DL-racemate. Thus, the whole molecule includes any combination of each stereoisomer of these amino acids, and the term “stereoisomer” is intended to include these combinations.

In the above formula (I), the “X ₁ ” group means a cysteine residue in the above formulas (IA) and (II-A), or the above formulas (IB) and (II-B). Means a group that binds to each amino group of an arginine residue. X group is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an acyl group having 1 to 6 carbon atoms, an alkoxycarbonyl group having 1 to 6 carbon atoms, an aralkyloxycarbonyl group, a natural amino acid residue or an unnatural amino acid. It can be a residue or a derivative thereof, a peptide residue consisting of a natural amino acid or a non-natural amino acid or a derivative thereof, or any group of the peptide residue derivative. As will be described later, in the disulfide exchange reaction of the present invention, the total charge of functional groups in the whole molecule is preferably 0 or more (preferably 1 or more) in neutral or basic, and the thiol group of the cysteine residue. It is preferable that the steric hindrance factor around is small. Therefore, the X ₁ group is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an acyl group having 1 to 4 carbon atoms, an alkoxycarbonyl group having 1 to 4 carbon atoms, a neutral or basic natural amino acid residue, or Those derivatives are preferable, and a hydrogen atom, a neutral or basic natural amino acid residue is more preferable.
Here, when X ₁ is referred to as an amino acid residue, it refers to the case where an amide (peptide) bond is formed at the carboxyl group moiety.

The “X ₂ ” group means a group bonded to the side chain or main chain amino group of the arginine residue in the above formulas (IA) and (II-A). X ₂ group is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an acyl group having 1 to 6 carbon atoms, an alkoxycarbonyl group having 1 to 6 carbon atoms, an aralkyloxycarbonyl group, a natural amino acid residue or a non-natural type It can be any group of amino acid residues or derivatives thereof, peptide residues consisting of natural amino acids or non-natural amino acids or derivatives thereof or peptide residue derivatives thereof. As described above, in the disulfide exchange reaction of the present invention, the total charge of the functional groups in the whole molecule is preferably 0 or more (preferably 1 or more), and steric inhibition around the thiol group of the cysteine residue. The factor is preferably small. Accordingly, the X ₂ group is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an acyl group having 1 to 4 carbon atoms, an alkoxycarbonyl group having 1 to 4 carbon atoms, a neutral or basic natural amino acid residue, or Those derivatives are preferable, and a hydrogen atom, a neutral or basic natural amino acid residue is more preferable. Moreover, it is preferable that Z group is a group couple | bonded with the amino group of the side chain of an arginine residue.
Here, when referred to as X ₂ is an amino acid residue refers to a case of forming the amide (peptide) bond at its carboxyl moiety.

The “Y” group means an arginine residue in the above formula (IA) and formula (II-A), or a cysteine residue in the above formula (IB) and formula (II-B). Means a group bonded to each carboxy group. Y group is a hydroxyl group, a hydrazino group, an alkoxy group having 1 to 6 carbon atoms, an appropriate 1 or 2 substituent (the substituent is an alkyl group having 1 to 6 carbon atoms, an acyl group having 1 to 6 carbon atoms) An amino group substituted with an alkoxycarbonyl group having 1 to 6 carbon atoms and an aralkyloxycarbonyl group), a natural amino acid residue or a non-natural amino acid residue, or a derivative thereof, a natural amino acid Alternatively, it may be a peptide residue consisting of a non-natural amino acid or a derivative thereof or any group of the peptide residue derivative. As described above, in the disulfide exchange reaction of the present invention, the total charge of the functional groups in the whole molecule is preferably 0 or more (preferably 1 or more), and steric inhibition around the thiol group of the cysteine residue. The factor is preferably small. Therefore, the Y group is a hydroxyl group, a hydrazino group, an alkoxy group having 1 to 4 carbon atoms, an appropriate 1 or 2 substituent (the substituent is an alkyl group having 1 to 4 carbon atoms, an alkyl group having 1 to 4 carbon atoms). An amino group substituted with an acyl group, an alkoxycarbonyl group having 1 to 4 carbon atoms, or an aralkyloxycarbonyl group), a neutral or basic natural amino acid residue, or a derivative thereof A hydroxyl group, a hydrazino group, an alkoxy group having 1 to 4 carbon atoms, an amino group, a neutral or basic natural amino acid residue is more preferable.
Here, when Y is called an amino acid residue, it means a case where an amide (peptide) bond is formed at the amino group moiety.

In one embodiment, combinations of X ₁ , X ₂ and Y groups include
X ₁ and X ₂ are each independently a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an acyl group having 1 to 4 carbon atoms, an alkoxycarbonyl group having 1 to 4 carbon atoms, a neutral or basic natural type An amino acid residue or a derivative thereof;
Y is a hydroxyl group, a hydrazino group, an alkoxy group having 1 to 4 carbon atoms, an appropriate 1 or 2 substituent (the substituent is independent of an alkyl group having 1 to 4 carbon atoms or an acyl group having 1 to 4 carbon atoms). An amino group substituted with a neutral or basic natural amino acid residue or a derivative thereof.

In another embodiment,
X ₁ and X ₂ are each independently a hydrogen atom or a neutral or basic natural amino acid residue;
Y is a hydroxyl group, a hydrazino group, an alkoxy group having 1 to 2 carbon atoms, an amino group, or a neutral or basic natural amino acid residue.

In yet another embodiment,
X ₁ is a hydrogen atom, or a neutral or basic natural amino acid residue;
Y is a hydroxyl group, a hydrazino group, an alkoxy group having 1 to 2 carbon atoms, an amino group, or a neutral or basic natural amino acid residue; and
The X ₂ group is a group that is bonded to the amino group on the side chain of the arginine residue and is a hydrogen atom.

である。 In yet another embodiment, the peptide of formula (I) has the formula:

It is.

  The term “alkyl group having 1 to 4 carbon atoms” or “alkyl group having 1 to 6 carbon atoms” means a linear or branched hydrocarbon group having 1 to 4 or 1 to 6 carbon atoms. Specific examples thereof include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, t-butyl group, pentyl group, isopentyl group, neopentyl group, n-hexyl group and the like. . A methyl group and an ethyl group are preferred.

  The term “acyl group having 1 to 4 carbon atoms” or “acyl group having 1 to 6 carbon atoms” means the alkyl group having 1 to 4 or 1 to 6 carbon atoms bonded to a carbonyl group (oxo group). Specific examples include acetyl group, propionyl group, butyryl group, isobutyryl group, valeryl group, isovaleryl group, pivaloyl group, n-pentylcarbonyl group, n-hexylcarbonyl group and the like. An acetyl group and a propionyl group are preferable.

  The term “C 1-4 alkoxy group” or “C 1-6 alkoxy group” means the alkyl group having 1 to 4 or 1 to 6 carbon atoms bonded to an oxygen atom. Includes a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, an isobutoxy group, a t-butoxy group, an n-pentoxy group, an isopentoxy group, a neopentoxy group, and an n-hexyloxy group. A methoxy group and an ethoxy group are preferable.

  The term “C1-C4 alkoxycarbonyl group” or “C1-C6 alkoxycarbonyl group” means the aforementioned C1-C4 or C1-C6 alkoxy bonded to a carbonyl group (oxo group). Specific examples include methoxycarbonyl group, ethoxycarbonyl group, n-propoxycarbonyl group, isopropoxycarbonyl group, n-butoxycarbonyl group, isobutoxycarbonyl tree, t-butoxycarbonyl group (Boc group), Examples include an n-pentoxycarbonyl group and an n-hexyloxycarbonyl group. A methoxy group, an ethoxy group, and a t-butoxycarbonyl group are preferable.

  The term “aralkyloxycarbonyl group” means an aralkyloxy group bonded to a carbonyl group (oxo group). Specific examples include benzyloxycarbonyl group (Z group), mono- or di-chloro-substituted benzyloxycarbonyl. Groups (including, for example, 2-, 3-, 4-, 2,4-, 2,6-, or 3,4-substituents). A benzyloxycarbonyl group is preferred.

  The term “amino group optionally substituted with 1 or 2 substituents” means an unsubstituted amino group or an amino group substituted with 1 or 2 substituents. As the said substituent, the said C1-C6 (C1-C4) alkyl group, the said C1-C6 (C1-C4) acyl group, the said C1-C6 (C1-C1) -4) a group selected from the group consisting of an alkoxycarbonyl group and an aralkyloxycarbonyl group. Specific examples include monomethylamino group, monoethylamino group, mono n-propylamino group, monoisopropylamino group, mono n-butylamino group, monoisobutylamino group, mono-t-butylamino group, monopentylamino group, Monocyclohexylamino group, dimethylamino group, diethylamino group, di-n-propylamino group, diisopropylamino group, di-n-butylamino group, diisobutylamino group, di-t-butylamino group, monoacetylamino group, monopropionylamino group , Monobutyrylamino group, mono (t-butyloxycarbonyl) amino group, mono (benzyloxycarbonyl) amino group and the like. Monomethylamino group, monoethylamino group, mono n-propylamino group, monoisopropylamino group, dimethylamino group, diethylamino group, monoacetylamino group, mono (t-butyloxycarbonyl) amino group, mono (benzyloxycarbonyl) An amino group is preferred.

  The term “natural amino acid residue” means a residue of a protein constituting amino acid, and is classified into a commonly used neutral, basic, or acidic amino acid residue. Specifically, neutral amino acid residues (for example, glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), proline (Pro), tryptophan (Trp), methionine (Met), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln)); basic amino acid residues (eg, arginine (Arg), lysine (Lys), histidine) (His)); and acidic amino acid residues such as aspartic acid (Asp) and glutamic acid (Glu). These amino acids include a D-form and an L-form, and the L-form is preferred from the viewpoint of availability. In the present specification, a single letter notation is appropriately used as the amino acid notation in addition to the three letter notation.

  As a neutral amino acid residue, Gly, Ala, Val, Leu, and Ile are preferable, and Gly or Ala is more preferable.

  As the basic amino acid residue, Arg and Lys are preferable.

  The term “non-natural amino acid residue” means an amino acid residue that is not a protein-constituting amino acid. The non-natural amino acid is naturally occurring or obtained by chemical synthesis, and is available from commercial owners (eg, Aldrich). Specific examples include ornithine (Orn), phenylglycine, 2-aminobutyric acid, β-alanine, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, homoserine, alloisoleucine, sarcosine, N-methylvaline, norvaline, etc. including. Ornithine (Orn) is preferred.

  The term “natural amino acid residue or derivative of a non-natural amino acid residue” means a derivative of the above “natural amino acid residue” or “non-natural amino acid residue”. One in which one or two hydrogen atoms on the group are substituted with an alkyl group or an acyl group, or one in which a hydroxyl group on a carboxyl group is substituted with an alkoxy group or an amino group.

  The term “a peptide residue consisting of a natural amino acid or a non-natural amino acid or a derivative thereof or a peptide residue derivative thereof” means two or more of the above-mentioned natural amino acid residues or non-natural amino acid residues or derivatives thereof. Means a peptide residue or a derivative thereof forming a peptide bond. The peptide residue includes a dipeptide or a tripeptide. In addition, the derivative means that one or two hydrogen atoms on the amino group of the constituent amino acid residue are alkyl as defined in the above-mentioned “derivative of natural amino acid residue or non-natural amino acid residue”. And those in which the hydroxyl group on the carboxyl group is substituted with an alkoxy group or an amino group.

The term “total of functional group charges in the whole molecule in neutral or basic” refers to the formula (I) (formula (II described below) under neutral or basic conditions for carrying out the disulfide exchange reaction in the present invention. The charge calculated by subtracting (sum of the number of negative charges on the negatively charged functional group) from (total number of positive charges on the positively charged functional group) in the entire molecule of means.
For example, positively charged functional groups include amino groups (which are present, for example, in the side chain of lysine or ornithine (including amino groups substituted by alkyls, etc.)), guanidino groups (which are, for example, arginine The number of positive charges of each of these groups is counted as +1. The negatively charged functional group includes a carboxyl group (which is present, for example, in the side chain of glutamic acid or aspartic acid), a sulfonic acid group (SO ₃ ⁻ ), etc., where the carboxyl group or sulfonic acid group The negative charge numbers are counted as -1 or -2, respectively. In addition, the neutral group is a group other than a functional group having a positive charge or a negative charge under the neutral or basic conditions (for example, an alkyl group, an aryl group, a hydroxyl group, a disulfide group). Means an amide group (—C (═O) NH ₂ —) (which is present, for example, in the side chain of asparagine or glutamine) or sulfonamide, which is a linking group between an amino group and a carboxyl group or a sulfonic acid group Also includes the group (—S (═O) ₂ NH ₂ —).

For example, in the case of the reduced α- (Arg-Cys-Gly) having the following structural formula, the positively charged functional group has one amino group and one guanidino group. The total number of positive charges on the base is calculated as +2. On the other hand, since there is one carboxy group for a negatively charged functional group, the total number of negative charges on the negatively charged functional group is calculated as -1. Therefore, the sum total of the charge of the functional group in the whole molecule is calculated as +1. It should be noted that the thiol (SH) group on the side chain of cysteine is not considered because it becomes a disulfide (SS) when a crossed disulfide bond is formed.
Similarly, in the case of reduced α- (Glu-Cys-Arg), since there is one amino group and one guanidino group for the positively charged functional group, it is on the positively charged functional group. The sum of the number of positive charges is calculated as +2. On the other hand, since there are two carboxy groups for the negatively charged functional group, the total number of negative charges on the negatively charged functional group is −2. calculate. Therefore, the sum total of the charges of the functional groups in the whole molecule is calculated as ± 0.
Similarly, in the case of reduced α- (Glu-Cys-Gly) (reduced α-glutathione), since there is one amino group for the positively charged functional group, the positively charged functional group The sum of the number of positive charges is calculated as +1. On the other hand, since there are two carboxy groups for the negatively charged functional group, the total number of negative charges on the negatively charged functional group is −2 And calculate. Therefore, the sum total of the charge of the functional group in this whole molecule is calculated as -1.

The compound represented by the formula I of the present invention is used as a disulfide exchange reaction accelerator that acts as a reducing reagent in the disulfide exchange reaction.
Here, the mechanism of the disulfide exchange reaction when the protein forms a three-dimensional structure (folding) is as described in detail in the definition of the term “disulfide exchange reaction accelerator” described later. Protein folding (three-dimensional structure formation) The rate-limiting step of the reaction is a nucleophilic reaction of the intramolecular thiol group (RS ⁻ ) to the crossed disulfide bond between the protein and the reagent of the present invention. Therefore, by placing a molecule (group) having a positively charged functional group around the sulfur atom of the crossed disulfide bond, the positive charge is localized around the sulfur atom, resulting in a thiol group in the protein having a negative charge. It can be expected that the reaction rate of the nucleophilic reaction by can be increased. Accordingly, molecules (groups) having groups that retain or increase the magnitude of the positive charge of the guanidino group on the arginine residue adjacent to the cysteine residue in the molecule of formula (I) are preferred.
Therefore, it is preferable that the sum of the charges of the functional groups in the whole molecule is ± 0 or more, preferably +1 or more. For example, a reduced form (Glu-Cys-Gly) (reduced glutathione) having a sum of charges of −1 In contrast, the reduced form (Glu-Cys-Arg) with a total charge of ± 0 and the reduced form (Arg-Cys-Gly) with a total charge of +1 are preferred, and the reduced form (Arg-Cys-Gly) is more preferred preferable.

  The term “peptide derivative” means a derivative of the peptide represented by formula (I), and specifically, one or two hydrogen atoms on the amino group of the constituent amino acid residues are replaced with another group (for example, An alkyl group or an acyl group) or a hydroxyl group on the carboxyl group substituted with another group (for example, an alkoxy group or an amino group).

  The term “salt” refers to a salt with a base (eg, a salt with an organic base (eg, ammonia, triethylamine, pyridine); an inorganic base (eg, an alkali metal (eg, sodium, potassium) or an alkaline earth metal (eg, Salt with acid (eg, formic acid, acetic acid, propionic acid, butyric acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, malic acid) , Mandelic acid, methanesulfonic acid, p-toluenesulfonic acid); salts with inorganic acids (for example, salts with hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, sulfurous acid), but are not limited thereto.

  The term “solvate” refers to hydrates; and organic solvents such as saturated hydrocarbons (eg, pentane, hexane), aromatic hydrocarbons (eg, benzene, toluene, xylene), halogenated hydrocarbons (eg, , Dichloromethane, chloroform, 1,2-dichloroethane), ethers (eg, diethyl ether, tetrahydrofuran, dioxane), ketones (eg, acetone), esters (eg, ethyl acetate), nitriles (eg, acetonitrile), Amines (eg, pyridine), acids (eg, formic acid, acetic acid), alcohols (eg, methanol, ethanol, isopropanol, t-butanol), or polar aprotic solvents (eg, N, N-dimethyl sulfoxide (DMSO) ), N, N-dimethylformamide (DMF) 1) or a mixture of two or more selected from, but not limited to. Hydrates and solvates with alcohols are preferred.

The term “disulfide exchange reaction promoter” means a reagent (agent) that promotes the formation or exchange reaction of an SS bond of a protein of interest having a disulfide (—S—S—) bond. The mechanism of the disulfide exchange reaction is as shown in the schematic diagrams 1 and 2 below. Here, when a protein having a disulfide bond forms a three-dimensional structure (sometimes referred to as “folding” in this specification and includes “refolding”), particularly when refolding an unfolded protein. Reformation of protein disulfide bonds is required. Such a reforming reaction requires a thiol-disulfide exchange reaction with a disulfide reagent. Here, dominates this disulfide exchange reaction, the protein S ^- and ^* RS-SR ^* kinetic factors intermolecular nucleophilic attack reactions with binding of oxidizing reagent, the protein in the S ^- by crossing disulfide This is considered to be an intramolecular nucleophilic attack reaction to the bond ( ^* RS-S) and a thermodynamic factor on the protein side.

Specifically, protein folding (stereostructure formation) by the disulfide exchange reaction reagent of the present invention crosses by the thiol group of the cysteine residue of the protein first causing an intermolecular nucleophilic attack on the disulfide bond of the oxidized reagent. A disulfide bond is formed (scheme 1 below). In Step 2 and Step 4, the intramolecular nucleophilic attack due to S ⁻ is likely to occur due to the positive charge of R ^* , so that the disulfide exchange reaction rate is increased. When the SS bond formed is a natural type, the folding reaction ends.
On the other hand, in the case of an incorrect binding mode, further SS exchange reaction proceeds as shown in the following schematic diagram 2. Here, in Step 3 ′, the intramolecular nucleophilic attack is likely to occur due to S ⁻ due to the positive charge of R ^* as in Step 2 and Step 4 in the above-described schematic diagram 1, so that the disulfide exchange reaction rate is increased.

Oxidizing reagent The disulfide exchange reaction of the present invention may further include an oxidizing reagent in addition to the reducing reagent represented by the formula (I). The oxidation reagent may be any generally known oxidation reagent, including, but not limited to, oxidized glutathione (GSSG), DTT _ox , Cys _{ox, and the} like. In the disulfide exchange reaction of the present invention, the monomer reducing reagent to be used can give an oxidizing reagent which is an oxidant derived from two molecules by oxygen present in the solution. The reaction in between is a reversible reaction.

で示されるペプチド、すなわち式（II−Ａ）および（II−Ｂ）で示されるペプチド、その立体異性体もしくはそのペプチド誘導体、またはそれらの塩もしくは溶媒和物から選択される少なくとも１種の化合物を含む。ここで、式（II）中の各基の定義は式（Ｉ）中の対応する各基の定義と同義である。また、ＡｒｇとＣｙｓとの結合がα結合またはγ結合であるかの点についても式（Ｉ）の場合に対応する。
また、当該式（II）で示されるペプチドは、システイン残基の側鎖にあるチオール基が酸化されていることから、本明細書中、「酸化型ペプチド」とも呼称する。 In addition, as an oxidizing reagent, each peptide represented by the above formula (I) (formula (IA) or (IB)) has a cysteine type in which a thiol group in the side chain of a cysteine residue is disulfide bonded. The following formula (II) forming an oxidant derived from two molecules:

At least one compound selected from the peptides represented by formula (II-A) and (II-B), stereoisomers or peptide derivatives thereof, or salts or solvates thereof: Including. Here, the definition of each group in Formula (II) is synonymous with the definition of each corresponding group in Formula (I). Further, the case of the formula (I) also corresponds to whether the bond between Arg and Cys is an α bond or a γ bond.
Further, the peptide represented by the formula (II) is also referred to as “oxidized peptide” in the present specification because the thiol group in the side chain of the cysteine residue is oxidized.

Like the compound of the above formula (I), in the above formula (II), Arg can form either Cys or an α bond or a γ bond. From the viewpoint of availability, α bond is preferable. A specific structural formula is shown below.

  In addition, the oxidizing reagent in the present invention includes a heterodimer obtained by combining reduced glutathione (GSH), which is a monomer of the oxidized glutathione, and the monomer compound of the formula I.

である。 In one embodiment, the peptide of formula (II) has the formula:

It is.

  In another embodiment, the disulfide exchange accelerator of the present invention includes the reduced form (Arg-Cys-Gly) and the oxidized form (Arg-Cys-Gly).

  When the disulfide exchange reaction accelerator of the present invention contains an oxidizing reagent, the blending ratio of the reducing reagent and the oxidizing reagent can be appropriately changed depending on the target protein, but the weight ratio is usually 0.01: 1 to 20: 1, preferably 1: 1 to 1:10.

  The ratio of the disulfide exchange reaction accelerator of the present invention used for carrying out the disulfide exchange reaction is not particularly limited. However, the reducing reagent is incorporated in a solution containing the target protein (for example, a refolding buffer). The concentration (total concentration) of (for example, reduced form of the peptide of formula (I)) and the oxidizing reagent (eg, oxidized form of the peptide of formula (II)) is usually 0.001 to 50 mg / mL, preferably 0. 0.01 to 10 mg / mL, more preferably 0.05 to 3 mg / mL.

  The concentration of the target protein contained in the solution containing the target protein (eg, refolding buffer) is usually 0.001 to 50 mg / mL, preferably 0.01 to 10 mg / mL, more preferably 0.05 to 3 mg / mL can be mentioned.

The term “stereostructure-forming agent” means an agent involved in a three-dimensional structure formation reaction of a protein. The three-dimensional structure-forming agent of the present invention includes, in addition to the disulfide exchange reaction accelerator of the present invention described above, that is, a compound of formula (I) as a reducing reagent and an oxidizing reagent (for example, a compound of formula (II), etc. One or more known additional protein conformation promoters may be included. Examples of the known further three-dimensional structure formation promoters for proteins include oxidation reagents (eg, oxidized glutathione (GSSG), DTT _ox , Cys _ox ), reducing reagents (eg, 2-mercaptoethanol, reduced glutathione (GSH)). , DTT _red , Cys _red ) and the like.

  The term “unfolded protein” means a protein in which the original three-dimensional structure of the protein is destroyed, that is, all proteins in a denatured state. For example, a protein produced by a genetic recombination technique and a protein completely unfolded using a denaturing agent are included.

  The term “refolding” means that the unfolded protein is again formed into a three-dimensional structure (folding), and is also called a regeneration method.

  The term “protein production method” is a generic term for a method for producing, regenerating or regenerating a protein, including a three-dimensional structure formation reaction of the protein including a disulfide exchange reaction of the protein.

  In the present invention, the target protein to be formed with a three-dimensional structure may be any protein having at least one disulfide bond, and may be natural or artificial (chemical synthesis method, fermentation method, gene recombination method), etc. Regardless, peptides, polypeptides, proteins, and complexes thereof (eg, complexes of (poly) peptides or proteins and compounds, complexes of (poly) peptides or proteins and saccharides, (poly) peptides or proteins) And metal complexes, (poly) peptides or proteins and coenzyme complexes, etc.). The type of protein is not limited and includes, for example, any of intracellular protein, extracellular protein, membrane protein, and nuclear protein. For example, lysozyme and ribonuclease are included.

  The target protein includes the target protein to be refolded, and is a recombinant produced by genetic engineering using a prokaryotic organism such as E. coli, a eukaryotic organism such as yeast, or a heterologous expression system such as a cell-free extraction system. It is. Since such recombinants are often obtained as insoluble and inactive aggregates, so-called inclusion bodies, the folding technique of the present invention can be suitably used.

  The unfolded protein at the time of refolding may be a protein that has been unfolded by any method, but from the viewpoint of the refolding effect, unfolded protein is preferably guanidine hydrochloride, urea, thiourea, or a combination thereof. It is a folded protein. More preferably, the concentration of guanidine hydrochloride, urea, thiourea or the total of these is usually 0.5 mol / L or more (for example, guanidine hydrochloride is 2 mol / L or more, urea is 3 to 4 mol / L or more). A protein unfolded in an aqueous solution. If the protein contains a disulfide bond in the molecule, in addition to the unfolding agent such as guanidine hydrochloride or urea, a mercapto reagent (reducing reagent such as 2-mercaptoethanol, dithiothreitol, cysteine, or thiophenol) may be used. ) May be unfolded protein.

  The protein targeted in the present invention is not particularly limited in its molecular weight, but can usually include peptides and proteins of about 1,000 to 10,000,00. From the viewpoint of the effect of forming a three-dimensional structure (particularly refolding), a protein having a molecular weight of 5,000 to 250,000 is preferred. According to the three-dimensional structure forming method using the three-dimensional structure forming agent of the present invention, since a high folding effect can be obtained, it is also effective for peptides having a molecular weight of less than 5,000. The molecular weight of the protein can be measured by a general gel electrophoresis method or the like.

II. 3D structure forming method The 3D structure forming method of the present invention is a method for producing a normal protein having activity by forming a 3D structure of a target protein, and treating the target protein in the presence of the 3D structure forming agent of the present invention described above. It has the process to perform.

  In the step of forming the three-dimensional structure, the proportion of the three-dimensional structure forming agent to be used is not limited, but as described above, the formula (I) blended in a solution containing the target protein (for example, a refolding buffer). As the concentration (total concentration) of the peptide of formula (II) and the like, the concentration is generally 0.01 to 100 mmol / L, preferably 0.05 to 10 mmol / L, more preferably 0.1 to 5 mmol / L. L can be mentioned. Here, the concentration of the target protein contained in the solution containing the target protein (for example, the refolding buffer) is usually 0.001 to 50 mg / mL, preferably 0.01 to 10 mg / mL, more preferably. Can be 0.05 to 3 mg / mL.

  The three-dimensional structure formation (folding) buffer used for three-dimensional structure formation (including refolding) is not particularly limited as long as it does not have a function and composition that cause the target protein to lose its function. For example, amine buffers such as Tris buffer, MES buffer, and tricine buffer, phosphate buffer, and various Good's buffers can be used. The buffer can be adjusted to pH 1-12, but is preferably in the range of pH 7-10, more preferably in the range of pH 7-9.

  A known reducing reagent (for example, reduced glutathione) and / or an oxidizing reagent (for example, oxidized glutathione) can be added to the buffer solution, and various additives can be added. Such additives include salts such as sodium chloride and calcium chloride; buffers such as citrate, phosphate and acetate; bases such as sodium hydroxide; acids such as hydrochloric acid and acetic acid; methanol, ethanol, Examples thereof include organic solvents such as propanol. The buffering agent includes a known reducing reagent (for example, reduced glutathione) and / or a known oxidizing reagent (for example, oxidized glutathione), or various additives, surfactants, pH adjusters, Alternatively, a protein stabilizer can be blended.

  Here, as the surfactant, any of a nonionic surfactant, a cationic surfactant, an anionic surfactant and an amphoteric surfactant can be used.

  Nonionic surfactants include, for example, higher alcohol alkylene oxide (hereinafter abbreviated as “AO”) adducts [higher alcohols having 8 to 24 carbon atoms (decyl alcohol, dodecyl alcohol, coconut oil alkyl alcohol, octadecyl alcohol and Oleyl alcohol etc.] ethylene oxide (hereinafter abbreviated as “EO”) 1-20 mol adduct, etc.], alkylphenol AO adduct having 6 to 24 carbons, polypropylene glycol EO adduct and polyethylene glycol PG Examples include adducts, pluronic surfactants, fatty acid AO adducts, polyhydric alcohol type nonionic surfactants, etc. Nonionic surfactants are preferred because they have less interaction with proteins. .

  Examples of the cationic surfactant include a quaternary ammonium salt type cationic surfactant and an amine salt type cationic surfactant. Examples of the anionic surfactant include ether carboxylic acid or a salt thereof, sulfate ester or ether sulfate ester and salt thereof, sulfonate, sulfosuccinate, fatty acid salt having a hydrocarbon group having 8 to 24 carbon atoms. , Acylated amino acid salts, and naturally occurring carboxylic acids and salts thereof (eg, chenodeoxycholic acid, cholic acid, deoxycholic acid, etc.). Examples of amphoteric surfactants include betaine-type amphoteric surfactants and amino acid-type amphoteric surfactants.

  When a surfactant is used, its content in a solution containing the target protein (for example, a refolding buffer) is usually 20% by weight or less, preferably 0.001 to 10% by weight, more preferably 0.01. -5% by weight can be mentioned. Basically, the critical micelle concentration or less of each surfactant is preferable.

  Examples of pH adjusters include Tris (trishydroxymethylaminomethane), HEPES (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid), and phosphate buffer (disodium monohydrogen phosphate + phosphoric acid 2). Monosodium hydrogen) and the like. In the present invention, the folding operation is performed at pH 7 to 10, preferably pH 7 to 9. For this reason, when adding a pH adjuster, the addition amount is adjusted to adjust to this pH range.

  Examples of protein stabilizers include reducing reagents, polyols, metal ions, chelating reagents, and amino acids or peptides. Here, as the reducing reagent, 2-mercaptoethanol, dithiothreitol, ascorbic acid, reduced glutathione, cysteine and the like; as the polyols, glycerin, glucose, sucrose, ethylene glycol, sorbitol and mannitol; as the metal ion Includes divalent metal ions such as magnesium ion, manganese ion and calcium ion. Examples of the chelating reagent include ethylenediaminetetraacetic acid (EDTA) and glycol etherdiamine-N, N, N ′, N′-4 acetic acid (EGTA). Examples of amino acids or peptides include arginine, polyarginine, and polylysine.

  When using a protein stabilizer, its content in a solution containing the target protein (for example, refolding buffer) is usually 10% by weight or less, preferably 0.001 to 10% by weight, more preferably 0.01. ˜1% by weight can be mentioned.

  In addition, the solution containing the target protein (for example, the refolding buffer) may further contain an unfolding agent, that is, a denaturing agent (for example, guanidine hydrochloride or urea).

  In the present invention, the step of treating the target protein in the presence of the three-dimensional structure forming agent includes the step of placing the protein and the three-dimensional structure forming agent under contact conditions, specifically, mixing both in a folding buffer and stirring. And the like. In addition, after that, standing for a certain period of time is included as necessary in order to further advance the formation of the three-dimensional structure. The standing time can be 1 to 50 hours, for example. Moreover, as temperature conditions, it can select suitably in the range of 0-100 degreeC according to the heat resistance of the protein made into object. Usually, it is the range of 4-50 degreeC.

III. Protein Production Method The protein production method of the present invention is a method including a step of forming a three-dimensional structure of a target protein using the above three-dimensional structure formation method (for example, refolding method), and is rephrased as a method of regenerating a normal protein. You can also

  The method for producing a protein of the present invention is not particularly limited as long as it has a step of treating the target protein in the presence of the above-described three-dimensional structure forming agent of the present invention. For example, in the following process, when a three-dimensional structure formation of a normal protein (for example, a modified protein prepared by genetic recombination) is performed, the step of unfolding the protein of (a) is not included, but the case of refolding Includes the step (a).

(A) a step of unfolding the protein;
(B) a step of treating the target protein (including the protein unfolded in the above step) in the presence of the disulfide exchange reaction accelerator (or three-dimensional structure forming agent) of the present invention to form a three-dimensional structure;
(C) A step of purifying the protein having the three-dimensional structure formed in the above step.

  When the target protein is a recombinant produced by genetic engineering using, for example, a heterologous expression system such as Escherichia coli, yeast, or a cell-free extraction system, the protein production method of the present invention includes the following (1) to (1) to It may be a method including the step (5).

(1) Culture process of protein-producing bacteria: A protein-producing bacterium such as Escherichia coli is cultured to produce a recombinant.
(2) Bacteria lysis step: The protein inclusion body is taken out from the protein-producing bacterium using a lysis agent or the like.
(3) Appropriate unfolding step: 0.5 mol / L or more of an unfolding agent (denaturing agent), and 20 mmol / L if necessary, in a suspension of the protein inclusion body (for example, 10 mg protein / mL) Add the following reducing reagent, stir lightly, heat to room temperature or about 50 ° C., and leave it for several minutes to 30 minutes. By this process, intramolecular or intermolecular disulfide bonds of proteins present in inclusion bodies are chemically reduced and cleaved.
(4) Folding step: adding the folding agent of the present invention to the unfolded protein suspension prepared in the above step (2) or (3) to dilute and lower the unfolding agent concentration, or The unfolded protein suspension is dialyzed to dilute and lower the unfolding agent concentration, and the folding agent of the present invention is added thereto for folding.
(5) Purification step: The target normal protein (refolded protein) is purified from the protein suspension obtained above using column chromatography or the like.

  The following bacterial cells can be exemplified as the protein-producing bacteria (host) in the above-mentioned (1) protein-producing bacteria culture step. Bacterial cells include Escherichia coli, Streptococci, Staphylococci, Escherichia, Streptomyces and Bacillus cells, eukaryotic cells; Cells and Aspergillus cells, insect cells; eg, Drosophilla S2 cells, Spodoptera Sf9 cells, animal cells; eg, CHO, COS, Hela, C127, 3T3, BHK, 293 and Bowes ( Bows) melanoma cells and plant cells.

The cDNA may be any method such as a recombination technique, a direct RT-PCR method, or a chemical synthesis method. An example of the recombination technique is shown below.
In the method for culturing a protein-producing bacterium in the above step (1), an expression vector containing cDNA encoding a target protein cell is (i) separating messenger RNA (mRNA) from the target protein-producing cell, and single-stranded from the mRNA. Then, double-stranded DNA is synthesized, and the complementary DNA is incorporated into a phage or plasmid. (Ii) The host is transformed with the obtained recombinant phage or plasmid, and after culture, the target DNA is transformed from the target DNA by hybridization with a DNA probe encoding a part of the target protein, or by immunoassay using an antibody. The cloned DNA to be produced can be excised and ligated downstream of the promoter in the expression vector. Thereafter, the host is transformed with the expression vector and cultured by an appropriate method. Culture is usually performed at 15 to 43 ° C. for 3 to 24 hours, and aeration and agitation can be added as necessary.

  As the lysis method employed in the lysis step (2) above, any of physical disruption by ultrasonic waves, treatment with a lytic enzyme such as lysozyme, treatment with a lytic agent such as a surfactant, and the like can be used. From the viewpoint of productivity, treatment with a lysing agent is preferred. In addition, from the viewpoint of not denaturing useful proteins, a lysing agent such as a quaternary ammonium type cationic surfactant whose counter ion is a carboxylic acid ion such as formic acid or acetic acid can be mentioned.

  Examples of the unfolding agent used in the unfolding step (3) include denaturing agents such as guanidine hydrochloride, urea and thiourea. Such modifiers can be used alone or in combination. When the protein is a protein containing a disulfide bond in the molecule, in addition to the denaturing agent, 2-mercaptoethanol, dithiothreitol, ascorbic acid, reduced glutathione, cysteine, etc. are added as a reducing reagent. Also good.

  In the purification step (5), examples of the filler used for column chromatography include silica, dextran, agarose, cellulose, acrylamide, and vinyl polymer. Commercially available products include the Sephadex series, Sephacryl series, Sepharose series (above Pharmacia), Bio-Gel series (Bio-Rad), and the like.

IV. Manufacturing method 1. Production of Reducing Reagent The compound (monomer) of the formula (I) of the present invention is commercially available or can be easily produced by a general organic chemistry method.

（ステップ１）
Ｎ末端保護（Ｐ^１）のＸ_１アミノ酸誘導体と、Ｃ末端保護（Ｐ^２）のＣｙｓ誘導体とを、汎用される脱水縮合剤（例えば、ジシクロヘキシルカルボジイミド（ＤＣＣ））を用いてカップリング反応する。
（ステップ２）
別途、Ｎ末端保護（Ｐ^３）のＡｒｇ誘導体と、Ｃ末端保護（Ｐ^４）のＹアミノ酸誘導体とを、上記脱水縮合剤を用いてカップリング反応する。
（ステップ３）
上記ステップ１および２で製造した各カップリング生成物の保護基Ｐ^２およびＰ^３をそれぞれ脱保護した後に、上記脱水縮合剤を用いてカップリング反応させ、次いで保護基Ｐ^１およびＰ^４を脱保護することにより、目的の式（I-A）の化合物を得ることができる。 An example of a general method for preparing the compound of formula (I) is shown below, but is not intended to be limited to this method. Moreover, the target compound can be obtained by using a general method for separation and resolution as required. For example, the production method of the compound of formula (IA) is shown in the following scheme 1.

(Step 1)
The X ₁ amino acid derivative of N-terminal protection (P ¹ ) and the Cys derivative of C-terminal protection (P ² ) are subjected to a coupling reaction using a commonly used dehydration condensing agent (for example, dicyclohexylcarbodiimide (DCC)).
(Step 2)
Separately, an Arg derivative with N-terminal protection (P ³ ) and a Y amino acid derivative with C-terminal protection (P ⁴ ) are subjected to a coupling reaction using the dehydration condensing agent.
(Step 3)
After deprotecting the protecting groups P ² and P ³ of each coupling product prepared in Steps 1 and 2, the coupling reaction is performed using the dehydrating condensing agent, and then the protecting groups P ¹ and P ⁴ are removed. By protecting, the target compound of formula (IA) can be obtained.

（ステップ１）
Ｎ末端保護（Ｐ^１）のＸ_１アミノ酸誘導体と、Ｃ末端保護（Ｐ^２）のＡｒｇ誘導体とを、汎用される脱水縮合剤（例えば、ジシクロヘキシルカルボジイミド（ＤＣＣ））を用いてカップリング反応する。
（ステップ２）
別途、Ｎ末端保護（Ｐ^３）のＣｙｓ誘導体と、Ｃ末端保護（Ｐ^４）のＹアミノ酸誘導体とを、上記脱水縮合剤を用いてカップリング反応する。
（ステップ３）
上記ステップ１および２で製造した各カップリング生成物の保護基Ｐ^２およびＰ^３をそれぞれ脱保護した後に、上記脱水縮合剤を用いてカップリング反応させ、次いで保護基Ｐ^１およびＰ^４を脱保護することにより、目的の式（Ｉ-B）の化合物を得ることができる。 A method for producing the compound of formula (IB) is shown in the following scheme 2.

(Step 1)
An X ₁ amino acid derivative of N-terminal protection (P ¹ ) and an Arg derivative of C-terminal protection (P ² ) are subjected to a coupling reaction using a commonly used dehydration condensing agent (for example, dicyclohexylcarbodiimide (DCC)).
(Step 2)
Separately, a Cys derivative with N-terminal protection (P ³ ) and a Y amino acid derivative with C-terminal protection (P ⁴ ) are subjected to a coupling reaction using the dehydration condensing agent.
(Step 3)
After deprotecting the protecting groups P ² and P ³ of each coupling product prepared in Steps 1 and 2, the coupling reaction is performed using the dehydrating condensing agent, and then the protecting groups P ¹ and P ⁴ are removed. By protecting, the target compound of formula (IB) can be obtained.

  The deprotecting group includes a deprotecting group commonly used in peptide synthesis. For example, an N-terminal protecting group (for example, a Boc (t-butyloxycarbonyl) group, a Z (benzyloxycarbonyl) group, Fmoc ( (9-fluorenylmethyloxycarbonyl) group), C-terminal protecting group (for example, OBzl (benzyl ester) group, OtBu (t-butyl ester) group), and amino acid side chain protecting group (Tos (tosyl) group) , Pbf (4-methoxytrityl) group, OcHex (cyclohexyl ester) group, MeBzl (methylbenzyl) group, Trt (trityl) group), but not limited thereto.

（ステップ１）
Ｎ末端をBoc保護および側鎖ＳＨ基をMeBzl保護したＣｙｓ誘導体と、Ｃ末端をBzl保護したＧｌｙ誘導体とを、有機溶媒（無水溶媒が好ましい）中、当量もしくは過剰量のＤＣＣの存在下、カップリング反応させ、次いでトリフルオロ酢酸（ＴＦＡ）処理によりBoc脱保護し、さらにトリエチルアミンを用いて中和することにより、Cys-Gly誘導体を得る。
（ステップ２）
上記ステップ１で得た生成物と、別途調製したArg誘導体とを、ステップ１と同様にＤＣＣの存在下、カップリング反応させ、次いでフッ化水素（ＨＦ）で処理することにより、脱保護（Tos、MeBzl、Bzl）して、目的のα-Arg-Cys-Glyを得る。 Next, a specific method for producing α-Arg-Cys-Gly (α-RCG) is shown in Scheme 3 below.

(Step 1)
A Cys derivative with Boc protection at the N-terminus and MeBzl-protection on the side chain SH group and a Gly derivative with Bzl-protection at the C-terminus are combined in an organic solvent (preferably anhydrous solvent) in the presence of an equivalent or excess amount of DCC. Ring reaction, followed by Boc deprotection by treatment with trifluoroacetic acid (TFA), followed by neutralization with triethylamine gives a Cys-Gly derivative.
(Step 2)
The product obtained in Step 1 above and the Arg derivative prepared separately were subjected to a coupling reaction in the presence of DCC in the same manner as in Step 1, and then treated with hydrogen fluoride (HF) to remove the deprotection (Tos , MeBzl, Bzl) to obtain the target α-Arg-Cys-Gly.

（ステップ１）
Ｎ末端をFmoc保護および側鎖ＳＨ基をTrt保護したＣｙｓ誘導体と、Ｃ末端をBzl保護したＧｌｙ誘導体とを、有機溶媒（無水溶媒が好ましい）中、当量もしくは過剰量のＤＣＣの存在下、カップリング反応させ、次いでピペリジン処理することによりFmocを脱保護して、Cys-Gly誘導体を得る。
（ステップ２）
上記ステップ１で得た生成物と、別途調製したArg誘導体とを、ステップ１と同様にＤＣＣの存在下、カップリング反応させ、次いでピペリジン処理、続いてＴＦＡ／ＴＩＳ（トリイソプロピルシラン）／水で処理後に、ＨＰＬＣ精製を行って、目的のα-Arg-Cys-Glyを得る。 Further, another production method of α-Arg-Cys-Gly (α-RCG) is shown in the following scheme 4.

(Step 1)
A Cys derivative in which the N-terminus is Fmoc-protected and the side chain SH group is Trt-protected, and a Gly derivative in which the C-terminus is Bzl-protected are combined in an organic solvent (preferably an anhydrous solvent) in the presence of an equivalent or excess amount of DCC. Fmoc is deprotected by ring reaction and then piperidine treatment to obtain a Cys-Gly derivative.
(Step 2)
The product obtained in Step 1 above and the Arg derivative prepared separately were subjected to a coupling reaction in the presence of DCC in the same manner as in Step 1, followed by piperidine treatment, followed by TFA / TIS (triisopropylsilane) / water. After the treatment, HPLC purification is performed to obtain the target α-Arg-Cys-Gly.

（ステップ１）
Ｎ末端をBoc保護および側鎖ＳＨ基をMeBzl保護したＣｙｓ誘導体と、Ｃ末端をBzl保護および側鎖グアニジノ基をTos保護したＡｒｇ誘導体とを、有機溶媒（無水溶媒が好ましい）中、当量もしくは過剰量のＤＣＣの存在下、カップリング反応させ、次いでトリフルオロ酢酸（ＴＦＡ）処理によりBoc脱保護し、さらにトリエチルアミンを用いて中和することにより、Cys-Arg誘導体を得る。
（ステップ２）
上記ステップ１で得た生成物と、別途調製したGlu誘導体とを、ステップ１と同様にＤＣＣの存在下、カップリング反応させ、次いでフッ化水素（ＨＦ）で処理することにより、脱保護（OcHex、Tos、MeBzl、Bzl）して、目的のα-Glu-Cys-Argを得る。 A specific method for producing α-Glu-Cys-Arg (α-ECR) is shown in Scheme 5 below.

(Step 1)
Cys derivative with Boc protection at the N-terminus and MeBzl side chain SH group and Arz derivative with Bzl protection at the C terminus and Tos protection at the side chain guanidino group in an organic solvent (preferably anhydrous solvent) The coupling reaction is carried out in the presence of an amount of DCC, followed by Boc deprotection by treatment with trifluoroacetic acid (TFA), and neutralization with triethylamine to obtain a Cys-Arg derivative.
(Step 2)
The product obtained in Step 1 above and a separately prepared Glu derivative were subjected to a coupling reaction in the presence of DCC in the same manner as in Step 1 and then treated with hydrogen fluoride (HF) to remove deprotection (OcHex , Tos, MeBzl, Bzl) to obtain the target α-Glu-Cys-Arg.

（ステップ１）
Ｎ末端をFmoc保護および側鎖ＳＨ基をTrt保護したＣｙｓ誘導体と、Ｃ末端を^tBu保護および側鎖グアニジノ基をPbf保護したＡｒｇ誘導体とを、有機溶媒（無水溶媒が好ましい）中、当量もしくは過剰量のＤＣＣの存在下、カップリング反応させ、次いでピペリジンを処理後して、Cys-Arg誘導体を得る。
（ステップ２）
上記ステップ１で得た生成物と、別途調製したGlu誘導体とを、ステップ１と同様にＤＣＣの存在下、カップリング反応させ、次いでピペリジン処理、続いてＴＦＡ／ＴＩＳ（トリイソプロピルシラン）／水で処理後に、ＨＰＬＣ精製を行って、目的のα-Glu-Cys-Argを得る。 Further, another production method of α-Glu-Cys-Arg (α-ECR) is shown in the following scheme 6.

(Step 1)
Cys derivative with Fmoc protection at the N-terminus and Trt protection at the side chain SH group and Arg derivative with ^t- Bu protection at the C-terminus and Pbf protection at the side chain guanidino group in an organic solvent (preferably anhydrous solvent) The coupling reaction is carried out in the presence of an excess amount of DCC, and then piperidine is treated to obtain a Cys-Arg derivative.
(Step 2)
The product obtained in Step 1 above and a separately prepared Glu derivative were subjected to a coupling reaction in the presence of DCC in the same manner as in Step 1, followed by piperidine treatment, followed by TFA / TIS (triisopropylsilane) / water. After the treatment, HPLC purification is performed to obtain the target α-Glu-Cys-Arg.

2. Production of Oxidizing Reagents Compounds of formula (II) of the present invention that can be used as oxidizing reagents (oxidized forms of two molecules of the compound of formula (I) (homotype)) or compounds of formula (I) of the present invention are known A hetero-type oxidant in combination with a reducing reagent (for example, GSH) can be easily produced by a general organic chemistry method.

  Specifically, the compound of the above formula (I) and / or a known reducing reagent is dissolved in a neutral or basic buffer, and then at room temperature in air for an appropriate time (from about 12 hours to about 1 It can be easily manufactured by leaving it to stand for oxidation reaction. The reaction concentration of the oxidizing reagent can be carried out in the reaction solvent at about 1 mmol / L to about 1 mol / L. In order to accelerate the production reaction, an oxidizing agent (for example, iron) or oxygen can be bubbled into the reaction solution.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Example 1
Production of oxidized α-Arg-Cys-Gly (oxidized α-RCG) Reduced α-Arg-Cys-Gly (10 mg, 0.030 mmol) was dissolved in 0.1 M Tris-HCl (pH 8.0) (150 μL). And allowed to stand in the dark for 10 days at room temperature. After completion of the reaction, the reaction solvent was distilled off under reduced pressure, and the residue was purified by HPLC to quantitatively obtain oxidized α-Arg-Cys-Gly. The obtained product was confirmed by various spectroscopic techniques.
MS (MH ^{+ 1} ) Theor .: 668.30; Found: 667.75.

(Example 2)
Disulfide bond reaction rate experiment Generally, protein disulfide bond formation reaction is slow, and it is necessary to reconstruct the wrong disulfide bond species as a reaction by-product. This is an important issue. Therefore, first, the reaction rate experiment of the disulfide bond reaction was conducted.

Here, glutathione is a tripeptide that promotes natural disulfide bond formation, which is essential for correct protein folding (stereostructure formation) in vivo, and is therefore widely used in in vitro research. Yes. However, optimizing conditions for correct disulfide bond formation in proteins using glutathione is difficult and often yields only low yields. This decrease in yield is primarily due to the formation of incorrect disulfide-bonded species within the protein. In other words, the correct disulfide bond selectivity in proteins by glutathione molecules is insufficient, and the development of molecular species aimed at selecting the correct disulfide bonds in proteins is desired.
In the present invention, in order to evaluate the ability of a folding promoting molecule, prouroguanylin containing three intramolecular disulfide bonds was employed, and the folding rate of prouroguanylin in various folding promoting molecules was used as an index.

Using prouroguanylin as an engineered protein, reduced glutathione (also referred to as “GSH”), oxidized glutathione (also referred to as “GSSG”) (Peptide Institute, Inc.), and reduced glutamilcysteinyl arginine (Reduced ECR), oxidized glutamyl cysteinyl arginine (oxidized ECR), and reduced arginyl cysteinyl glycine (reduced RCG), oxidized arginyl cysteinyl glycine (oxidized RCG) folding agent As a function.
(1) Experimental procedure A large amount of prouroguanylin is expressed from an E. coli expression system, and the resulting intracellular inclusion bodies (inactive aggregates) are reduced with denaturing denaturing reagents (8 M urea, 20 mM dithiothreitol, 0.1 M Tris). -HCl (pH 8.0)), followed by reduction-modified prouroguaniline obtained by reverse-phase HPLC (Hitachi High-Tech) and reverse-phase column Cosmosil 5C18-AR-2 (8 x 250) mm) (Nacalai Tesque Co., Ltd.) and dried under reduced pressure to prepare reduced modified prouroguanylin in a vacuum. A folding reaction solution comprising the following composition containing reduced / oxidized glutathione, reduced / oxidized ECR, and reduced / oxidized RCG as a folding agent is added to the resulting reduced modified prouroguanylin (20 nmol) at room temperature. We evaluated the kinetic effects of disulfide bond formation in the folding reaction. In addition, a spectrophotometer (wavelength 220 nm) attached to the HPLC was used for identification and quantification of the folding intermediate of prouroguanylin in the reaction.
<Folding reaction solution>
(1) 2 mM GSH, 1 mM GSSG, 0.1 M Tris-HCl (pH 8.0) (control)
(2) 2 mM reduced ECR, 1 mM oxidized ECR, 0.1 M Tris-HCl (pH 8.0)
(3) 2 mM reduced RCG, 1 mM oxidized RCG, 0.1 M Tris-HCl (pH 8.0)

(2) Experimental Results FIGS. 1 to 3 show the results of investigating the formation rate of disulfide formation in the refolding of prouroguanylin for various folding agents.
The main problem in the refolding reaction with the correct disulfide bond formation in the protein is how the reaction intermediate (such as molecular species with different disulfide bonds) transitions from the denatured state to the natural state (active state). It disappears quickly and takes a natural structure.

1 and 2, R is prouroguanylin having no disulfide bond in the reduced denaturation state, I is prouroguanylin having the wrong disulfide bond of the refolding intermediate, and prourolog having the correct disulfide bond of the natural type. Aniline is shown as N. Here, each retention time is confirmed by a standard substance (reference: Biochemistry 1998, 37, 8498-8507; THE JOURNAL OF BIOLOGICAL CHEMISTRY 2000, 275, 25155-25162). Specifically, R is about 28.0 minutes, I is a group of molecular species between about 24.0 and about 26.5 minutes, and N is about 25.5 minutes.
Therefore, in FIG. 1, in the presence of reduced / oxidized glutathione, the presence of an intermediate in refolding was observed until about 30 minutes after refolding.

  RCG and ECR were created by substituting arginine, which is widely used for the purpose of increasing solubility in refolding, into the lead chemical species. FIG. 2 shows the formation of disulfide bonds of prouroguanylin during refolding using reduced / oxidized ECR. Here, the folding intermediate I disappeared within 10 minutes after refolding. This shows a faster refolding effect than glutathione. Furthermore, FIG. 3 shows disulfide bond formation of prouroguanylin during refolding using reduced / oxidized RCG. As a result, disappearance of I as a folding intermediate was observed within 5 minutes. This is correct disulfide bond formation much earlier than the existing glutathione redox system, and in order to obtain other refolding effects, circular dichroism measurement is also used in protein structure formation and compared with the reaction in glutathione redox system did.

(Experimental example 3)
As described above in Example 2, disulfide bond formation in folding using reduced / oxidized RCG showed much faster disulfide bond formation than the existing glutathione redox system. However, not only correct disulfide bonds but also correct secondary structure formation such as α-helix and β-sheet is essential for protein structure formation, which is important for the construction of physiologically active expression structures. Therefore, in this experimental example, when a correct secondary structure is formed, prouroguanylin is adopted as a protein having a spectrum characteristic to the wavelength of the α helix (222 nm), as in Experimental Example 1. The function as a folding agent was compared by comparing the secondary structure formation by reduced / oxidized glutathione and reduced / oxidized RCG. The various chemical species used were the same compounds as those used in Experimental Example 2.

(1) Experimental procedure As in Experimental Example 2, a folding reaction solution comprising the following composition containing the obtained reduced modified prouroguanylin (2 nmol) as a folding agent: reduced / oxidized glutathione and reduced / oxidized RCG. And the kinetic effects of secondary structure formation in the folding reaction at room temperature were estimated. The secondary structure formation rate of prouroguanylin during folding was estimated by fixing the wavelength at 222 nm using a circular dichroism spectrometer (JASCO Corporation).
<Folding reaction solution>
(1) 2 mM GSH, 1 mM GSSG, 0.1 M Tris-HCl (pH 8.0, 300 μL) (control)
(2) 2 mM reduced RCG, 1 mM oxidized RCG, 0.1 M Tris-HCl (pH 8.0, 300 μL)

(2) Experimental results FIG. 4 shows the results of measuring the rate effect of disulfide formation in the folding of prouroguanylin for various folding agents.
As a result, it is clear that reduced / oxidized RCG is far faster than reduced / oxidized glutathione (Kgsh = 1.29 × 10 ^-3 s ^-1 ) until complete secondary structure formation in folding. (Krcg = 3.69 × 10 ⁻³ s ⁻¹ ). In other words, compared to reduced / oxidized glutathione, reduced / oxidized RCG can provide superior differences in two points: 1) disulfide bond formation and 2) secondary structure formation, and a folding agent with excellent folding speed. Met.

(Experimental example 4)
As described above in Examples 2 and 3, RCG is a molecular species that accelerates the rate of disulfide bond formation and secondary structure formation in folding by about 3 times compared to glutathione. In addition, protein folding under a high concentration of protein is considered difficult (reference: Biosci Biotechnol Biochem. (2000). 64, 1159-65; Journal of Biotechnology 130 (2007) 153-160). Therefore, in order to compare the function as a folding agent, lysozyme (Seikagaku Corporation) in the folding of reduced / oxidized glutathione (GSH / GSSG), reduced / oxidized ECR, and reduced / oxidized RCG. The folding yield was determined. The various chemical species used were the same compounds as those used in Experimental Example 2.

(1) Experimental operation
A reducing denaturing reagent (8 M urea, 20 mM dithiothreitol, 0.1 M Tris-HCl (pH 8.3)) was added to 5 mg lysozyme, and the mixture was allowed to stand in a thermostat at 40 ° C. for 3 hours. Then, it dialyzed with respect to 10 mM HCl, the density | concentration was measured by the light absorbency in 220 nm by HPLC, and it was lyophilized | freeze-dried. 1 mL of a folding solution having the following composition was added to 0.1 to 1.6 mg of the resulting reduced modified lysozyme and allowed to stand at room temperature for 48 hours.
<Folding reaction solution>
(1) 2 mM GSH, 1 mM GSSG, 0.1 M Tris-HCl (pH 8.0) (control)
(2) 2 mM reduced ECR, 1 mM oxidized ECR, 0.1 M Tris-HCl (pH 8.0)
(3) 2 mM reduced RCG, 1 mM oxidized RCG, 0.1 M Tris-HCl (pH 8.0)

  After 48 hours, the resulting reaction solution was centrifuged under the conditions of 15,000 × g and 4 ° C., and the collected soluble fraction (centrifugated supernatant) was subjected to HPLC to evaluate the folding yield. In addition, by subjecting to HPLC, it is possible to evaluate whether the produced protein has formed a natural disulfide bond. The lysozyme folding yield was calculated as a relative value (%) when the concentration of lysozyme in the reduced denaturation state was 100% by weight, based on the absorbance at 220 nm in HPLC.

(2) Experimental results FIG. 5 shows the results of the folding yield of lysozyme for various folding agents. As a result, the yield of reduced / oxidized ECR did not change compared to reduced / oxidized glutathione, but the yield of reduced / oxidized RCG was 130, 0.2, 0.4, 0.8, 1.6 mg / mL. Increased 131, 171, 185%. That is, reduced / oxidized RCG was a folding agent that was superior in folding yield compared to reduced / oxidized glutathione. Conventionally, a method of increasing the yield of folding by adding an additive of several hundred mM such as arginine is known. The present invention is more effective than the folding yields known in the conventional methods, and is considered to be an advantageous folding reagent because it shows an effect with a small amount (several mM) of the folding agent.

(Experimental example 5)
By employing lysozyme as described above in Example 2, the effect of RCG on the folding yield was shown. Therefore, in order to improve versatility, in this experimental example, as in Experimental Example 3, prouroguanylin is used as a protein, and reduced / oxidized glutathione (GSH / GSSG) and reduced / oxidized RCG are folded. The functions as agents were compared in terms of yield. The various chemical species used were the same compounds as those used in Experimental Example 2.

(1) Experimental operation 1 mL of a folding solution having the following composition was added to 0.1 to 0.4 mg of the reduced modified prouroguanylin prepared by the method shown in Experimental Example 2, and left at room temperature for 48 hours.
<Folding reaction solution>
(1) 2 mM GSH, 1 mM GSSG, 0.1 M Tris-HCl (pH 8.0) (control)
(2) 2 mM reduced RCG, 1 mM oxidized RCG, 0.1 M Tris-HCl (pH 8.0)

(2) Experimental results
The result in the folding yield in RCG and glutathione is shown in FIG. As a result, compared to reduced / oxidized glutathione, the yield of reduced / oxidized RCG increased by 114, 143% at 0.2, 0.4 mg / mL. That is, reduced / oxidized RCG was a folding agent that was superior in folding yield compared to reduced / oxidized glutathione. The folding yield of prouroguanylin by RCG was increased compared to glutathione, similar to lysozyme. This is generally considered to increase the yield of folding of many proteins.

(Experimental example 6)
Using lysozyme and prouroguanylin as proteins, the yield of the folding effect was confirmed to be higher than that of oxidized / reduced glutathione of oxidized RCG / reduced RCG. In prouroguanylin, oxidized RCG / reduced RCG accelerated the folding rate about 3 times compared to oxidized / reduced glutathione. Therefore, we considered that RCG could promote the ability to select the correct disulfide bonds in proteins, and evaluated the ability of RCG molecules to exchange disulfide bonds in proteins. Here, prouroguanylin can determine the cross-linking position of intramolecular disulfide bonds by enzymatic treatment with arginyl endopeptidase, and can accurately evaluate the abundance ratio of the produced disulfide isomers. The correct disulfide bond selectivity can be evaluated. Therefore, by examining the production ratio of the natural form in the experiment that suppresses the disulfide exchange reaction in the protein without the coexistence of the reducing reagent, that is, the structure formation experiment of prouroguanylin using only the oxidized reagent, It was decided to evaluate the selectivity of type disulfide bonds. For this purpose, only the oxidized form, i.e., oxidized glutathione or oxidized RCG was employed as the folding promoting reagent (no reduced form coexisted), and the prouroguanylin folding reaction was performed.

(1) Experimental operation 1 mL of a folding solution having the following composition was added to reduced modified prouroguanylin (20 nmol) prepared by the method shown in Experimental Example 2, and left at room temperature for 48 hours.
<Folding reaction solution>
(1) 1 mM GSSG, 0.1 M Tris-HCl (pH 8.0) (control)
(2) 1 mM oxidized RCG, 0.1 M Tris-HCl (pH 8.0)

(2) Experimental results FIG. 7 shows the results (by HPLC) of the ability to select correct disulfide bonds in proteins in folding with oxidized RCG and oxidized glutathione. The correct disulfide bond is indicated as N, and the incorrect disulfide bond is indicated as I. Each holding time is as described in Example 2 above. As a result, in the case of oxidized glutathione (A), I: N was 6: 5, whereas in the case of oxidized RCG (B), I: N was 5:11. In other words, oxidized RCG increased the rate of correct natural disulfide bond formation by a factor of about 2 compared to oxidized glutathione. That is, oxidized RCG is a folding agent that has an excellent ability to form a correct disulfide bond compared to oxidized glutathione.

  By using the disulfide exchange reaction accelerator of the present invention, the correct three-dimensional structure formation of a natural protein can be obtained quickly and with good yield, and the industrial utility value is high.

Claims (12)

Formula (I) as a reducing reagent:

[Where:
X ₁ and X ₂ are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an acyl group having 1 to 6 carbon atoms, an alkoxycarbonyl group having 1 to 6 carbon atoms, an aralkyloxycarbonyl group, a natural amino acid A residue or a non-natural amino acid residue or a derivative thereof, a peptide residue consisting of a natural amino acid or a non-natural amino acid or a derivative thereof, or a peptide residue derivative thereof;
Y is a hydroxyl group, a hydrazino group, an alkoxy group having 1 to 6 carbon atoms, an appropriate 1 or 2 substituent (the substituent is an alkyl group having 1 to 6 carbon atoms, an acyl group having 1 to 6 carbon atoms, An amino group substituted with an alkoxycarbonyl group having 1 to 6 carbon atoms and an aralkyloxycarbonyl group independently selected from natural amino acid residues or non-natural amino acid residues or derivatives thereof, natural A peptide residue or a peptide residue derivative thereof consisting of a type amino acid or a non-natural amino acid or a derivative thereof;
Arg is α- or γ-bonded to Cys;
The total charge of functional groups in the whole molecule is 0 or more in neutral or basic]
Or at least one compound selected from the stereoisomers or peptide derivatives thereof, or salts or solvates thereof; and, optionally, formula (II) as an oxidizing reagent:

[Where:
X ₁ , X ₂ and Y are as defined above;
Arg is α- or γ-bonded to Cys, where
The total charge of functional groups in the whole molecule is 0 or more in neutral or basic]
At least one compound selected from a peptide represented by the following, a stereoisomer or peptide derivative thereof, or a salt or solvate thereof:
A disulfide exchange reaction accelerator. X ₁ and X ₂ are each independently a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an acyl group having 1 to 4 carbon atoms, an alkoxycarbonyl group having 1 to 4 carbon atoms, or a neutral or basic natural amino acid. Residues or derivatives thereof;
Y is a hydroxyl group, a hydrazino group, an alkoxy group having 1 to 4 carbon atoms, an appropriate 1 or 2 substituent (the substituent is an alkyl group having 1 to 4 carbon atoms, an acyl group having 1 to 4 carbon atoms, An amino group substituted with an alkoxycarbonyl group having 1 to 4 carbon atoms and an aralkyloxycarbonyl group independently selected from the group consisting of neutral or basic natural amino acid residues or derivatives thereof,
The disulfide exchange reaction accelerator according to claim 1.   The disulfide exchange reaction accelerator according to claim 1 or 2, wherein the natural amino acid residue is a basic amino acid residue selected from the group consisting of L-Arg and L-Lys.   The disulfide exchange accelerator according to any one of claims 1 to 3, wherein the total charge of functional groups in the whole molecule is 1 or more in neutral or basic.   The disulfide exchange reaction accelerator according to any one of claims 1 to 4, wherein Arg is α-bonded to Cys.   The disulfide exchange reaction accelerator according to any one of claims 1 to 5, further comprising at least one other oxidizing reagent.   The disulfide exchange reaction accelerator according to any one of claims 1 to 6, wherein a weight ratio of the reducing reagent to the oxidizing reagent is 0.01 to 20.   Further, according to any one of claims 1 to 7, further comprising an additive selected from the group consisting of a buffer solution, salts, buffer agents, bases, acids, organic solvents, surfactants, pH adjusters and protein stabilizers. 2. The disulfide exchange reaction accelerator according to 1. (A) the reducing reagent, and optionally the additive selected from the group consisting of the buffer solution, salts, buffers, bases, acids, organic solvents, surfactants, pH adjusters, protein stabilizers;
(B) the reducing reagent, and an additive selected from the group consisting of the buffer, salts, buffers, bases, acids, organic solvents, surfactants, pH adjusters, and protein stabilizers as appropriate;
The disulfide exchange reaction accelerator according to any one of claims 1 to 8, wherein the disulfide exchange reaction accelerator is contained in the same container form or in a separately packaged form.   A protein three-dimensional structure-forming agent comprising the disulfide exchange reaction accelerator according to any one of claims 1 to 9 and, optionally, at least one further protein three-dimensional structure formation promoter.   The protein three-dimensional structure forming agent according to claim 10, wherein the three-dimensional structure formation of the protein is refolding.   A protein comprising the step of forming a protein three-dimensional structure using the disulfide exchange reaction accelerator according to any one of claims 1 to 9 or the protein three-dimensional structure forming agent according to claim 10 or 11. Production method.

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