JP2009073815A - Refolding agent for protein, and method for producing the protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a refolding agent for refolding a protein apt to coagulate, and to provide a method for producing the resultant protein. <P>SOLUTION: The refolding agent (I) essentially comprises (A) a phosphorus-containing compound having a group of general formula (1) and (B) a nonionic surfactant. Alternatively, the refolding agent (II) comprises, (C) an oxycarbonyl group-containing compound having one or more groups selected from a carboxy group, a carboxylate anionic group and an ester group and (B) a nonionic surfactant. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a protein refolding agent and a production method, and more particularly to a refolding agent used for unwinding an unfolded protein into an active protein structure and a method for producing the protein.

  Elucidation and analysis of protein functions and structures are extremely important, for example, directly related to disease treatment and drug discovery. For this reason, various proteins are synthesized and produced by various methods, their structures are examined, and action mechanisms and roles in the living body are elucidated. Now, it is well known that the functions of proteins are determined not only by the sequence and chain length of the amino acids constituting them, but also by the ordered three-dimensional structure (higher order structure) taken by them.

Industrially, with the development of genetic engineering, various proteins have been prepared in large quantities as recombinants and used in a wide variety of industries such as pharmaceutical manufacturing, food processing, and clinical diagnosis. With the development of vector technology, technology that produces a large amount of target protein in Escherichia coli and yeast cells can be easily and reproducibly executed with few resources.
However, many of the proteins expressed in recombinants are unordered in their three-dimensional structure, their higher-order structures are not controlled, and often form small particle granules called inclusive inclusion bodies (inclusion bodies). . Therefore, in the E. coli production process, the inclusion body is unwound (unfolded), the higher-order structure is prepared, and the protein is converted into a soluble protein with an ordered three-dimensional structure, that is, the inclusion body is unfolded and then refolded. It is necessary to (rewind).

  This type of refolding is an extremely important technique that can be applied not only to proteins produced by E. coli and yeast, but also to the regeneration of proteins inactivated due to certain causes such as thermal history. Therefore, this refolding has been extensively studied mainly in the dialysis method and dilution method, and various methods have been proposed. Most of them have a low refolding rate and some limited proteins. Often only favorable results were obtained accidentally (especially for low molecular weight specific proteins), and this refolding is now general and universal, applicable to various proteins Moreover, it is not an efficient and economical method with a high refolding rate.

  The inventor has already found a refolding method capable of obtaining high productivity by using a specific phosphate or carboxylate as a refolding agent (Patent Documents 1 and 2).

However, in this method, a protein having a relatively simple structure such as lipase is unwound, but when used for a protein containing an SS bond in a molecule such as lysozyme, the SS bond is correctly corrected during refolding. Frequently, proteins are aggregated without being recombined into an incorrect three-dimensional structure. As a result, the yield of active protein may be extremely reduced.
JP 2007-126391 A JP 2007-145801 A

  Accordingly, it is a problem to provide a refolding agent and a protein production method that hardly cause protein aggregation even when used for a protein containing an S—S bond in the molecule, and to improve the yield of active protein. It is.

As a result of intensive studies to solve the above problems, the present inventor has reached the present invention.
That is, the present invention is an unfolded protein refolding agent comprising a phosphorus-containing compound (A) having a group represented by the general formula (1) and a nonionic surfactant (B) as essential components. An unfolded protein refolding agent having an oxycarbonyl group-containing compound having one or more groups selected from the group consisting of a carboxyl group, a carboxylate anion group and an ester group ( A refolding agent (II) comprising C) and a nonionic surfactant (B) as essential components; and a method for producing a protein comprising a step of refolding a protein with the refolding agent (I) or (II). .

  When the refolding agent of the present invention is used, protein aggregation during refolding can be suppressed more than before, and many active proteins can be obtained.

  The refolding agent of the present invention is an unfolded protein refolding agent, and is roughly classified into a refolding agent (I) comprising a phosphorus-containing compound (A) and a nonionic surfactant (B) as essential components. And a refolding agent (II) having an oxycarbonyl group-containing compound (C) and a nonionic surfactant (B) as essential components. That is, a phosphorus-containing compound (A) composed of a compound (A) having a group represented by the following general formula (1), a refolding agent (I) having a nonionic surfactant (B) as essential components, a carboxyl A refolding agent (II) comprising, as essential components, a compound (C) having one or more groups selected from the group consisting of a group, a carboxylate anion group and an ester group, and a nonionic surfactant (B).

  The refolding agent (I) of the first invention of the present application comprises a phosphorus-containing compound (A) and a nonionic surfactant (B) as essential components, and this phosphorus-containing compound (A) has the following general formula (1) ).

P in the general formula (1) represents a phosphorus atom, and examples of the phosphorus-containing compound (A) include inorganic phosphoric acid and its salt (A1); alkyl phosphate ester and its salt (A2); and sugar phosphate ester and its salt Salt (A3) etc. are mentioned.
One type of phosphorus-containing compound (A) may be used, or two or more types may be used in combination.

Inorganic phosphoric acid and its salt (A1) include phosphoric acid, hypophosphorous acid, phosphorous acid, hypophosphoric acid, pyrophosphoric acid, pyrophosphorous acid, metaphosphoric acid, tripolyphosphoric acid, polyphosphoric acid, and salts thereof. Can be mentioned.
Salts include alkali metal salts (sodium salt, potassium salt, lithium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, barium salt, etc.), ammonium salt, amine salt (primary amine salt, secondary amine salt). Tertiary amine salts), and quaternary ammonium salts (such as tetraalkylammonium salts).

  As a salt, when (A1) contains a plurality of acid groups, all of the acid groups may be a salt, or a part of the acid groups may be a salt.

  Specific examples of the salt of (A1) include phosphoric acid monohydrogen disodium salt, phosphoric acid dihydrogen monosodium salt, phosphoric acid monohydrogen dipotassium salt, phosphoric acid dihydrogen monopotassium salt, ammonium phosphate salt, Phosphate salts such as tetramethylammonium phosphate, tetraethylammonium phosphate, triethylamine phosphate and triethanolamine phosphate; sodium phosphite, potassium phosphite, ammonium phosphite salt, tetraphosphite tetra Phosphites such as methylammonium salt, tetraethylammonium phosphite, triethylamine phosphite and triethanolamine phosphite; sodium pyrophosphate, potassium pyrophosphate, tetramethylammonium pyrophosphate, pyrophosphoric acid Tetraethylammonium salt, triethylamine pyrophosphate And including pyrophosphates such as pyrophosphate triethanolamine salts.

Examples of the alkyl phosphate ester and its salt (A2) include alkyl phosphate ester (A21), alkyl pyrophosphate ester (A22), and alkyltripolyphosphate ester (A23) having an alkyl group having 1 to 12 carbon atoms. .
Examples of the alkyl phosphate ester (A21) having an alkyl group having 1 to 12 carbon atoms include monoalkyl phosphate esters, dialkyl phosphate esters, and trialkyl phosphate esters. Monoalkyl phosphates include methyl phosphate, ethyl phosphate, propyl phosphate, 2-propyl phosphate, butyl phosphate, 2-butyl phosphate, t-butyl phosphate, pentyl phosphate, hexyl phosphate Examples include esters, cyclohexyl phosphates, octyl phosphates, nonyl phosphates, decyl phosphates and the like. Examples of the dialkyl phosphate ester include diesters of the above alkyl phosphate ester, and examples of the trialkyl phosphate ester include triesters of the above alkyl phosphate ester.

  Examples of the alkyl pyrophosphate ester (A22) having an alkyl group having 1 to 12 carbon atoms include monoalkyl pyrophosphate esters, dialkyl pyrophosphate esters, and trialkyl pyrophosphate esters. Examples of monoalkyl pyrophosphate esters include methyl pyrophosphate ester, ethyl pyrophosphate ester, propyl pyrophosphate ester, 2-propyl pyrophosphate ester, butyl pyrophosphate ester, 2-butyl pyrophosphate ester, t-butyl pyrophosphate ester, and pentyl. Pyrophosphate ester, hexyl pyrophosphate ester, cyclohexyl pyrophosphate ester, octyl pyrophosphate ester, nonyl pyrophosphate ester, decyl pyrophosphate ester and the like can be mentioned. Examples of the dialkyl pyrophosphate ester include the above-described alkyl pyrophosphate ester diester, and examples of the trialkyl pyrophosphate ester include the above-described alkyl pyrophosphate ester triester.

  Examples of the alkyltripolyphosphate (A23) having an alkyl group having 1 to 12 carbon atoms include monoalkyltripolyphosphate, dialkyltripolyphosphate, and trialkyltripolyphosphate. Monoalkyltripolyphosphates include methyltripolyphosphate, ethyltripolyphosphate, propyltripolyphosphate, 2-propyltripolyphosphate, butyltripolyphosphate, 2-butyltripolyphosphate, t-butyltripolyphosphate, pentyl Examples include tripolyphosphate, hexyltripolyphosphate, cyclohexyltripolyphosphate, octyltripolyphosphate, nonyltripolyphosphate, decyltripolyphosphate. Examples of the dialkyltripolyphosphate include diesters of the above alkyltripolyphosphate, and examples of the trialkyltripolyphosphate include triesters of the above alkyltripolyphosphate.

Examples of the salts (A21) to (A23) include the same alkali metal salts, alkaline earth metal salts, ammonium salts, amine salts and quaternary ammonium salts as the salts mentioned in the above (A1).
As a salt, when there are a plurality of acid groups contained in (A21) to (A23), all of the acid groups may be a salt, or a part of the acid group may be a salt.

  Sugar phosphates and their salts (A3) include adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and cyclic adenosine monophosphate (c-AMP) and their sodium Salt, potassium salt, triethylamine salt, triethanolamine salt and the like can be mentioned.

Of the phosphorus-containing compound (A), (A1) and (A3) are preferred from the viewpoint of reducing protein aggregation and / or improving the yield of active protein, and more preferred are phosphoric acid, pyrophosphoric acid, polyphosphoric acid, and adenosine. Triphosphate, adenosine diphosphate, adenosine monophosphate and their salts.

  In the refolding step, the concentration of (A) in the system is preferably 0.1 to 6 mol / L, more preferably 0.2 to 6 from the viewpoint of reducing protein aggregation and / or improving the yield of active protein. Used in mol / L.

Here, the system refers to a state in which water, protein, denaturant and refolding agent are mixed, and the concentration in the system refers to the refolding agent relative to the volume of the mixed solution of water, protein, denaturant and refolding agent. The same applies in the following description.
The denaturant refers to a drug for unraveling the three-dimensional structure of a misfolded protein, and a substance that cleaves the hydrogen bond and disulfide bond of the protein is used. Specifically, 6 mol / L guanidine hydrochloride aqueous solution and 8 mol / L urea aqueous solution are mentioned. Mercaptoethanol, dithiothreitol or the like may be added and used in order to completely break the disulfide bond.

  As the nonionic surfactant (B), which is another essential component in the refolding agent (I) of the present invention, higher alcohol alkylene oxide (hereinafter abbreviated as AO) adduct (B1), carbon AO adduct (B2) of alkylphenol having an alkyl group of 6 to 24, polypropylene glycol ethylene oxide (hereinafter abbreviated as EO) adduct (B3), polyethylene glycol propylene oxide (hereinafter abbreviated as PO). An adduct (B4), a fatty acid AO adduct (B5), a polyhydric alcohol type nonionic surfactant (B6) and the like are included.

  Here, AO refers to alkylene oxide, and specifically includes ethylene oxide (EO), propylene oxide (PO), butylene oxide, and the like.

  As the AO adduct (B1) of higher alcohols, EO 1-20 mol adducts of higher alcohols having 8 to 24 carbon atoms (decyl alcohol, dodecyl alcohol, palm oil alkyl alcohol, octadecyl alcohol, oleyl alcohol, etc.) Examples thereof include PO1-40 mol adducts of higher alcohols having 8 to 24 carbon atoms. From the viewpoint of protein aggregation and / or protein yield improvement, an EO 1-20 mol adduct of a higher alcohol having 8 to 24 carbon atoms is preferable, and EO 18 mol of oleyl alcohol is more preferable.

  As the AO adduct (B2) of alkylphenol, EO1-40 mol adduct of C7-C36 alkylphenol (nonylphenol, cetylphenol, etc.), and PO1-40 mol adduct of C7-C36 alkylphenol are also used. Etc. From the viewpoint of protein aggregation and / or protein yield improvement, an EO 1 to 40 mol adduct of alkylphenol having 7 to 36 carbon atoms is preferable, and Triton-X100, Triton-X300, and the like are easily available.

  Examples of the polypropylene glycol EO adduct (B3) include a pluronic surfactant, and an EO 2-40 mol adduct of polypropylene glycol having 10 to 100 propylene oxide repeating units. From the viewpoint of preventing protein aggregation and / or improving the yield of protein, polypropylene glycol EO 10 mol adduct having 20 to 50 propylene oxide repeating units and polypropylene glycol EO 25 mol addition having 20 to 50 propylene oxide repeating units Things are preferred.

  Examples of the polyethylene glycol PO adduct (B4) include polyethylene glycol PO 2-20 mol adducts having 10 to 100 ethylene oxide repeating units. From the viewpoint of preventing protein aggregation and / or improving protein yield, A polyethylene glycol PO10 molar adduct having 10 to 50 ethylene oxide repeating units is preferred.

Examples of the fatty acid AO adduct (B5) include EO1 to 25 mol adducts of higher fatty acids having 8 to 24 carbon atoms (such as lauric acid, stearic acid, and oleic acid). From the viewpoint of preventing protein aggregation and / or improving the yield of protein, lauric acid EO 10 mol adduct, stearic acid EO 20 mol adduct and oleic acid EO 20 mol adduct are preferred.
Note that (B5) is handled as (C) when used in combination with the oxycarbonyl group-containing compound (C), and (B5) in other cases.

  Examples of the polyhydric alcohol type nonionic surfactant (B6) include EO adducts of alcohols having two or more hydroxyl groups in the molecule. From the viewpoint of preventing protein aggregation and / or improving protein yield, sorbitan alkyl ester EO 6 to 40 mol adduct and glycerin alkyl ester EO 2 to 40 mol adduct are preferable, and sorbitan alkyl ester EO 6 to 40 mol adduct is more preferable. TWEEN20, TWEEN40, TWEEN60, TWEEN80, etc. are easily available.

Among these, from the viewpoint of preventing protein aggregation, higher alcohol AO adduct (B1) and polyhydric alcohol AO adduct (B6) are preferable, and (B1) is more preferable.
Preferable specific examples include adducts of 1 to 20 moles of EO of higher alcohols having 8 to 24 carbon atoms (oleyl alcohol, decyl alcohol, dodecyl alcohol, coconut oil alkyl alcohol, octadecyl alcohol, etc.); sorbitan alkyl ester EO adducts and the like. It is done.

  In the refolding step using the refolding agent (I), from the viewpoint of preventing protein aggregation, the concentration of the nonionic surfactant (B) in the system is preferably 0.001 to 10% by weight, more preferably. Is 0.01 to 1% by weight.

  In the refolding step, the ratio of (A) concentration (% by weight) to (B) concentration (% by weight) in the system is from 0.05 to 50,000 (% by weight / weight) from the viewpoint of preventing protein aggregation. %), More preferably 1 to 5,000 (wt% / wt%).

The refolding agent (II) of the second invention of the present application comprises an oxycarbonyl group-containing compound (C) and a nonionic surfactant (B) as essential components, and the oxycarbonyl group-containing compound (C) is a carboxyl group. , A compound having one or more groups selected from the group consisting of a carboxylate anion group and an ester group.
This oxycarbonyl group-containing compound (C) has an oxycarbonyl group (—COO—) in the molecule.

The oxycarbonyl group-containing compound (C) includes a compound (C1) having at least one carboxyl group (—COOH) or at least one carboxylate anion group (—COO ⁻ ) in the molecule, and The compound (C2) which has at least 1 ester group (-COOR) is mentioned. In addition, oxycarboxylic acids such as lactic acid having these oxycarbonyl group and hydroxyl group simultaneously are also included in the oxycarbonyl group-containing compound (C) of the present invention.

  As the compound (C1) having at least one carboxyl group or carboxylate anion group in the molecule, a normal carboxylic acid (C11) having no polyoxyalkylene group in the molecule, a polyoxyalkylene group-containing carboxylic acid (C12 ); And their salts.

  Examples of the carboxylic acid (C11) include an aliphatic carboxylic acid (C111) having 1 to 36 carbon atoms (including the carbon atom of the carbonyl group, the same shall apply hereinafter), an aromatic carboxylic acid having 7 to 36 carbon atoms (C112), and the like. It is done.

  Examples of the aliphatic carboxylic acid (C111) include aliphatic saturated monocarboxylic acids having 1 to 36 carbon atoms (formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, verargonic acid, Lauric acid, myristic acid, stearic acid, behenic acid, 2-ethylhexanoic acid, etc.); aliphatic unsaturated monocarboxylic acids having 3 to 36 carbon atoms (acrylic acid, methacrylic acid, oleic acid, etc.); Aliphatic hydroxy monocarboxylic acids (glycolic acid, lactic acid, tartaric acid, gluconic acid, etc.); aliphatic saturated dicarboxylic acids having 2 to 36 carbon atoms (oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, Suberic acid, azelaic acid, sebacic acid and the like) and their monoalkyl esters; aliphatic unsaturated dicarboxylic acids having 4 to 36 carbon atoms (maleic acid) Inic acid, fumaric acid, itaconic acid, etc.); and trivalent or higher (preferably 3-12 valent) aliphatic polyvalent carboxylic acids (citric acid, butanetetracarboxylic acid, cyclopentanetetracarboxylic acid, ethylenediaminetetraacetic acid, Diethylenetriaminepentaacetic acid); and the like.

  As aromatic carboxylic acid (C112), C7-C36 aromatic monocarboxylic acid (benzoic acid, cinnamic acid, hydroxybenzoic acid, etc.); C8-C36 aromatic dicarboxylic acid (phthalic acid, isophthalic acid) , Terephthalic acid, etc.); C9-36 aromatic tricarboxylic acids and tetracarboxylic acids (trimellitic acid, pyromellitic acid, etc.); aromatic hydroxycarboxylic acids (salicylic acid, etc.) and the like.

  As the carboxylic acid (C11), a partial alkyl (having 1 to 12 carbon atoms as the alkyl group) ester (monomethyl oxalate, monomethyl succinate, maleate) of the above dicarboxylic acid or a trivalent or higher polyvalent carboxylic acid. Monomethyl acid, monoethyl oxalate, monomethyl citrate, dimethyl citrate, etc.).

  Examples of the polyoxyalkylene group-containing carboxylic acid (C12) include ether carboxylic acid compounds represented by the general formula (2).

^{R 1 -O- (R 2 O)} p-R 3 COOH (2)
(In the formula, R ¹ represents a hydrocarbon group having 1 to 36 carbon atoms, R ² represents an alkylene group having 2 to 4 carbon atoms, R ³ represents an alkylene group having 1 to ³ carbon atoms, and p represents an integer of 1 to 50. .)

R ¹ is a hydrocarbon group having 1 to 36 carbon atoms, which is a methyl group, an ethyl group, an n-propyl group, a 2-propyl group, an n-butyl group, a 2-butyl group, a t-butyl group, a pentyl group, Examples include n-hexyl group, cyclohexyl group, octyl group, nonyl group, decyl group, dodecyl group and the like.
A part of hydrogen of R ¹ may be substituted with a hydroxyl group.
R ² is an alkylene group having 2 to 4 carbon atoms, and examples thereof include an ethylene group, a 1,2-propylene group, a 1,3-propylene group, a 1,2-butylene group, and a 1,4-butylene group. .
R ³ is an alkylene group having 1 to 3 carbon atoms, and examples thereof include a methylene group, an ethylene group, a 1,3-propylene group, and a 1,2-propylene group. p is an integer of 1 to 50, and is preferably 1 to 20, more preferably 1 to 10, and particularly preferably 1 to 4, from the viewpoint of improving the protein yield.

Specific examples of ether carboxylic acids include polyoxyethylene hexyl ether acetic acid, polyoxyethylene octyl ether acetic acid, polyoxyethylene nonyl ether acetic acid, polyoxyethylene decyl ether acetic acid, polyoxyethylene dodecyl ether acetic acid, polyoxypropylene hexyl ether acetic acid. , Polyoxypropylene octyl ether acetic acid, polyoxypropylene nonyl ether acetic acid, polyoxypropylene decyl ether acetic acid, polyoxypropylene dodecyl ether acetic acid and the like.
The ether carboxylic acid can be produced by adding an alkylene oxide to a higher alcohol and then reacting with a monochlorocarboxylic acid.

As the compound (C1) having at least one carboxylate anion group in the molecule, a salt of a carboxylic acid (C11) or a polyoxyalkylene group-containing carboxylic acid (C12) is also included.
Examples of the salt include alkali metal salts, alkaline earth metal salts, ammonium salts, amine salts, and quaternary ammonium salts similar to the salts mentioned in (A1).

  Specific examples of the salt of the carboxylic acid (C11) include aliphatic saturated monocarboxylates having 1 to 36 carbon atoms (sodium formate, ammonium formate, guanidinium formate, sodium acetate, ammonium acetate, guanidinium acetate, sodium propionate). , Ammonium propionate, guanidinium propionate, sodium butyrate, sodium caproate, etc.); aliphatic unsaturated monocarboxylic acids having 3 to 36 carbon atoms (such as sodium acrylate); aliphatic hydroxymonocarboxylic acids having 3 to 36 carbon atoms Salts (sodium glycolate, sodium lactate, ammonium lactate, guanidinium lactate, sodium tartrate, potassium tartrate, ammonium tartrate, guanidinium tartrate, sodium gluconate, etc.); aliphatic saturated dicarboxylates having 2 to 36 carbon atoms (oxalic acid) Thorium, sodium succinate, sodium glutarate, etc.); aliphatic unsaturated dicarboxylates having 4 to 36 carbon atoms (such as sodium maleate); partial alkyl esters of the above dicarboxylic acids or trivalent or higher polyvalent carboxylic acids Salts (monomethyl sodium oxalate, monomethyl sodium succinate, monomethyl potassium maleate, monoethyl sodium oxalate, monomethyl sodium citrate, dimethyl sodium citrate, etc.).

  Specific examples of the salt of the polyoxyalkylene group-containing carboxylic acid (C12) include polyoxyethylene hexyl ether sodium acetate, polyoxyethylene octyl ether sodium acetate, polyoxyethylene nonyl ether sodium acetate, polyoxyethylene decyl ether sodium acetate, Examples include sodium polyoxyethylene dodecyl ether acetate, sodium polyoxypropylene hexyl ether acetate, sodium polyoxypropylene octyl ether acetate, sodium polyoxypropylene nonyl ether acetate, sodium polyoxypropylene decyl ether acetate, sodium polyoxypropylene dodecyl ether acetate It is done.

  Among the oxycarbonyl group-containing compound (C) of the present invention, as the compound (C2) having at least one ester group in the molecule, carboxylic acid alkyl ester (C21), addition of alkylene oxide of carboxylic acid A thing (C22) etc. are contained.

Examples of the carboxylic acid constituting the carboxylic acid alkyl ester (C21) include the same carboxylic acids as exemplified as the compound (C1) having at least one carboxyl group or carboxylate anion group in the molecule.
The alkyl group constituting the alkyl ester group includes methyl group, ethyl group, n-propyl group, 2-propyl group, n-butyl group, 2-butyl group, t-butyl group, pentyl group, and n-hexyl. And a linear or branched aliphatic alkyl group having 1 to 12 carbon atoms such as a group, a cyclohexyl group, an octyl group, a nonyl group, a decyl group and a dodecyl group.

  Specific examples of the carboxylic acid alkyl ester (C21) include monocarboxylic acid alkyl esters (such as methyl formate, methyl acetate, ethyl formate, ethyl acetate, n-butyl acetate, methyl stearate and n-butyl stearate); dicarboxylic acid Dialkyl esters (dimethyl oxalate, dimethyl succinate, dimethyl maleate, diethyl oxalate, diethyl succinate, dimethyl adipate, etc.); all carboxyl groups of trivalent or higher polyvalent aliphatic polycarboxylic acids are esterified Such as trimethyl citrate and tetramethyl ethylenediaminetetraacetate.

Examples of the alkylene oxide adduct (C22) of carboxylic acid include adducts of 1 to 50 mol of alkylene oxide having 2 to 4 carbon atoms of carboxylic acid mentioned in the above (C1).
Examples of the alkylene oxide include ethylene oxide, propylene oxide, and butylene oxide.

  Specific examples of carboxylic acid alkylene oxide adducts include monocarboxylic acid EO adducts (formic acid EO 1-10 mol adduct, acetic acid EO 1-10 mol adduct, propionic acid EO 10 mol adduct, butyric acid EO 1-10 mol addition. Products, caproic acid EO 2-20 mol adduct, lauric acid EO 1-20 mol adduct, etc.); oxycarboxylic acid EO adduct (glycolic acid EO 1-20 mol adduct, lactic acid EO 1-20 mol adduct, tartaric acid PO1- EO adducts of aliphatic polycarboxylic acids (oxalic acid EO 1-20 mol adduct, malonic acid EO 1-20 mol adduct, succinic acid EO 1-20 mol adduct, glutaric acid EO1-20 Mole adduct, citric acid EO 1-20 mol adduct, maleic acid EO 1-20 mol adduct, etc.); aromatic polycarboxylic acid with EO Things (EO1～30 mol adduct of phthalic acid, terephthalic acid EO1～30 mol adduct); and the like.

  Among these compounds (C), from the viewpoint of preventing protein aggregation and / or improving the yield of the protein, (C1) and its salt are preferable, more preferably aliphatic carboxylic acid (C11) and its salt, particularly preferably These are aliphatic carboxylic acids having 1 to 8 carbon atoms (for example, formic acid, acetic acid, propionic acid, lactic acid, tartaric acid) and salts thereof.

  In the refolding step, the concentration of (C) in the system is preferably 0.001 to 6 mol / L, more preferably 0.01 to 6 from the viewpoint of preventing protein aggregation and / or improving the protein yield. Used in mol / L.

  The nonionic surfactant (B) as an essential component in the refolding agent (II) of the present invention is the same as the nonionic surfactant (B) shown in the above-mentioned refolding agent (I). The preferred ones are the same.

  In the refolding step using the refolding agent (II), the concentration of (B) in the system is preferably 0.001 to 10% by weight, more preferably 0.01 to 1% from the viewpoint of preventing protein aggregation. % By weight.

  In the refolding step, the ratio of the concentration of (C) to the concentration of (B) in the system is preferably 0.01 to 60,000 (wt% / wt%), more preferably from the viewpoint of preventing protein aggregation. 1 to 6,000 (wt% / wt%).

The refolding agent (I) or (II) of the present invention may contain water, a salt, a buffering agent, glutathione and the like as necessary.
When water is contained, the content is not particularly limited, but it is 100 to 10 from the viewpoint of operability with respect to the total weight of the compounds (A) and (B) or the total weight of (C) and (B). 1,000% by weight is preferable, and more preferably 200 to 5,000% by weight.
Examples of the salt include sodium chloride, ammonium chloride, potassium chloride and the like, and the content thereof is the total weight of the compounds (A) and (B) or the total weight of (C) and (B). From the viewpoint of improving the yield, 1 to 10% by weight is preferable, and more preferably 1 to 3% by weight.
Examples of the buffer include Tris buffer, HEPES buffer, and the like. The content of the buffer is the total weight of the compounds (A) and (B) or the total weight of (C) and (B). From the viewpoint of improvement, it is preferably 1 to 30% by weight, and more preferably 1 to 10% by weight.
In the case of containing glutathione, the content is 0.001 to 0 in terms of improving the protein yield with respect to the total weight of the compounds (A) and (B) or the total weight of (C) and (B). 0.5 wt% is preferable, and 0.01 to 0.1 wt% is more preferable.

  The target protein is preferably a protein having an S—S bond in the molecule (for example, lysozyme) because the effect of the present invention is remarkable. In this case, the refolded protein is combined with guanidine hydrochloride or urea, Furthermore, the protein is unfolded by adding a reducing agent such as 2-mercaptoethanol, dithiothreitol, cystine, and thiophenol.

Proteins in the present invention can be classified into a protein (P1) containing a disulfide bond in the molecule and a protein (P2) containing no disulfide bond in the molecule.
Examples of the protein (P1) containing a disulfide bond in the molecule include lysozyme, β-lactoglobulin, alkaline phosphatase, ribonuclease, trypsin, chymotrypsin, interferon, insulin, natriuretic peptide and antibody.
Examples of the protein (P2) containing no disulfide bond in the molecule include lipase, phospholipase, and a peptide composed of 2 to 10 amino acids.
Among these, as the protein to be the subject of the present invention, a protein (P1) containing an S—S bond in the molecule is preferable because the effects of the present invention are remarkable. The number of S—S bonds in a molecule greatly affects the aggregation of proteins, and generally the larger the number, the easier the aggregation. The number of S—S bonds in the protein molecule to be the subject of the present invention is preferably 1 to 10, and more preferably 2 to 4. Specific examples include lysozyme (4), β-lactoglobulin (2-3), trypsin (2), and the like.

The molecular weight of the protein in the present invention is 1,000 to 300,000, and preferably 10,000 to 250,000 from the viewpoint of ease of refolding. Here, the molecular weight refers to the molecular weight estimated by SDS gel electrophoresis.
In general, there is a correlation between the molecular weight and the difficulty of refolding, and it is said that refolding becomes extremely difficult when the protein has a large molecular weight (molecular weight of about 10,000 or more). Since the refolding method of the present invention has an excellent refolding effect, it is a very effective method even for high molecular weight proteins having a molecular weight of 10,000 or more.

  The “refolding step” in the present invention is a step of stirring and mixing the protein and the refolding agent, and thereafter, it is allowed to stand for a certain period of time as necessary in order to further advance the refolding. When stirring and mixing the protein and the refolding agent, it is preferable to stir and mix until almost no heterogeneous portion is eliminated. The standing time is not particularly limited, but is, for example, 1 to 50 hours. Moreover, the temperature at the time of a process process is although it does not specifically limit, For example, it is 4-30 degreeC.

The step of refolding using the refolding agent of the present invention may further use a pH adjuster (D) and a protein stabilizer (E) in the step of treating with the refolding agent.
Examples of the pH adjuster (D) include Tris (N-tris (hydroxymethyl) methylaminoethanesulfonic acid), HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), and the like.

  In addition, when using a conventional phosphate buffer (for example, sodium dihydrogen phosphate + aqueous hydrochloric acid solution, or sodium dihydrogen phosphate + sodium hydroxide aqueous solution), the phosphate buffer is a phosphate. Therefore, it overlaps with (A) in the present invention. However, the upper limit of the addition concentration of the phosphate buffer used for the purpose of the conventional pH adjustment is 0.05 mol / L at the highest, and the phosphorus compound (A) as the refolding agent of the present invention and the conventional The concentration at the time of use is clearly different from the phosphate buffer whose pH is to be adjusted. The conventional phosphate buffer is handled as the phosphorus-containing compound (A) in the present invention.

  In general, the refolding operation is performed at pH 7 to 8, and the amount of the pH adjuster (D) used is not particularly limited as long as it is adjusted within this range, but the weight is (A) or (C). On the other hand, from the viewpoint of preventing protein aggregation, the content is preferably 20% by weight or less, more preferably 0.001 to 20% by weight, and further preferably 0.01 to 20% by weight.

Examples of the protein stabilizer (E) include a redox agent, a polyol, a metal ion, and a chelating reagent.
Examples of the redox agent include 2-mercaptoethanol, dithiothreitol, ascorbic acid, oxidized glutathione, reduced glutathione, cysteine, and cystine.
Examples of the polyol include glycerin, glucose, sucrose, ethylene glycol, sorbitol and mannitol.
Examples of metal ions include divalent metal ions such as magnesium ions, manganese ions, and calcium ions.
Examples of the chelating reagent include ethylenediaminetetraacetic acid (EDTA) and glycol etherdiamine-N, N, N ′, N′-4 acetic acid (EGTA).

  The amount of the protein stabilizer (E) used is preferably 10% by weight or less, more preferably from the viewpoint of preventing protein aggregation and / or improving the protein yield, relative to the weight of (A) or (C). 0.001 to 10% by weight, then more preferably 0.01 to 5% by weight.

In the refolding method of the present invention, the protein concentration in the system is preferably 0.2 to 30 mg / mL, more preferably 0.2 to 20 mg / mL, and particularly preferably 0.25 to 5 mg / mL. If it is 0.2 mg / mL or more, it is preferable from the viewpoint of protein production efficiency, and if it is 30 mg / mL or less, the viscosity in the system is rarely excessively high, and thus production efficiency does not decrease.
Moreover, the usage-amount (weight) of (A) or (C) with respect to the weight of protein is 5-2,000 parts with respect to 1 part of protein from a viewpoint of protein aggregation prevention and / or a protein yield improvement. Is more preferable, and more preferably 10 to 1,000 parts.

The protein production method of the present invention is a protein production method including a step of refolding with the above refolding agent.
Since the protein obtained by the method for producing a protein of the present invention is obtained by using the above refolding agent, there is little aggregation during the refolding step, and the yield is remarkably improved.
Examples of the protein production method of the present invention include a production method according to the following sequence of steps.
(1) Protein cultivation process: Enzyme or recombinant protein is cultured in a protein producer such as E. coli.
(2) Lysis process: The inclusion body in the protein producing body is taken out by using a lysing agent or the like.
(3) Unfolding step: 0.5 mol / L or more unfolding agent and 20 mmol / L or less reducing agent are added to an inclusion body suspension (for example, 10 mg protein / mL), and the mixture is gently stirred and left at room temperature for several hours. .
(4) Refolding step: Add (I) or (II) to the unfolded protein solution, stir lightly, and leave at room temperature overnight to perform refolding.
(5) Separation / removal step: The target refolded protein is separated from the suspension by column chromatography or the like.

Examples of the protein producer in the protein culturing step (1) include the following bacterial cells, Escherichia and Bacillus.
Bacterial cells include streptococci, staphylococci, escherichia, streptomyces and bacillus cells, fungal cells such as yeast cells and aspergillus Aspergillus cells, insect cells: eg Drosophila S2, Spodoptera Sf9 (Spodoptera Sf9) cells, animal cells: eg CHO, COS, Hela, C127, 3T3, BHK, 293 and Bowes melanoma cells A plant cell etc. are mentioned.
As Escherichia (Escherichia), Escherichia coli (E. coli) K12DH1 [Proc. Natl. Acad. Sci. 160 (1968)], JM103 (see Nucleic Acids Research, Vol. 9, 309 (1981)), JA221 (Journal of Molecular Biology). ) 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (196) See year)], C600 [Genetics (Genetics) 39 vol, see 440 pp (1954)], MM294 [Nature (Nature) 217 Vol see 1110 pages to (1968)] and the like.
Examples of Bacillus include Bacillus subtilis MI114 (see Gene, 24, 255 (1983)), 207-21 (Journal of Biochemistry 95, 87). Page (1984)].

As a recombinant protein culturing method, an expression vector containing cDNA encoding the target protein is:
(I) A messenger RNA (mRNA) is isolated from the target protein-producing cell, a single-stranded cDNA and then a double-stranded DNA are synthesized from the mRNA, and the complementary DNA is incorporated into a phage or a plasmid.
(Ii) The host is transformed with the obtained recombinant phage or plasmid, and after culture, contains the DNA of interest by hybridization with a DNA probe encoding a part of the protein of interest or by immunoassay using an antibody Isolate phage or plasmid.
(Iii) It can be produced by cutting out the desired cloned DNA from the recombinant DNA and ligating the cloned DNA or a part thereof 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 in the lysis process, any of physical disruption by ultrasound, treatment with a lytic enzyme such as lysozyme, treatment with a lysis agent such as a surfactant, etc. can be used. A treatment with a bacteriolytic agent described in JP-A-2006-320313 is particularly preferred from the viewpoint of not denatured useful proteins.

  Examples of the packing material used for column chromatography in the protein separation / removal process include silica, dextran, agarose, cellulose, acrylamide, vinyl polymer, etc., and commercially available products such as Sephadex series, Sephacryl series, Sepharose series (above, Pharmacia series). And Bio-Gel series (Bio-Rad) are available.

  The following examples further illustrate the invention, but the invention is not limited thereto.

Production Example 1 (Preparation of unfolding solution)
An unfolding solution for unfolding the protein once was 6 mol / L guanidine hydrochloride, 10 mmol / L dithiothreitol, 150 mmol / L sodium chloride, 50 mmol / L Tris buffer (pH = 8), Each was added to 1 mmol / L sodium ethylenediaminetetraacetate and dissolved in sterilized water.

Example 1 (Preparation 1 of refolding agent)
In a 20 mL sterilized vial, 1.0 mol / L sodium acetate, 0.10 wt% oleyl alcohol ethylene oxide 18 mol adduct (“Emalmin 180” manufactured by Sanyo Chemical Industries), 50 mmol / L Tris buffer Solution (pH = 8), 300 micromol / L oxidized glutathione, 150 mmol / L reduced glutathione (both manufactured by Wako Pure Chemical Industries, Ltd.), respectively, dissolved in sterilized water, and refolding agent (R- 1) was prepared.

Example 2 (Preparation of Refolding Agent 2)
In Example 1, in place of using 0.10 wt% oleyl alcohol ethylene oxide 18 mol adduct, 0.10 wt% sorbitan monooleate EO adduct (“TWEEN 80” manufactured by Wako Pure Chemical Industries, Ltd.) was used. In the same manner as in Example 1, a refolding agent (R-2) was prepared.

Example 3 (Preparation of Refolding Agent 3)
In Example 1, a refolding agent was carried out in the same manner as in Example 1 except that 1.0 mol / L disodium hydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) was used instead of 1.0 mol / L sodium acetate. (R-3) was prepared.

Example 4 (Preparation of Refolding Agent 4)
A refolding agent (R-4) was prepared in the same manner as in Example 1 except that 8.0 mol / L sodium acetate was used instead of 1.0 mol / L sodium acetate.

Example 5 (Preparation of Refolding Agent 5)
A refolding agent (R-5) was prepared in the same manner as in Example 1 except that 0.013 mol / L sodium acetate was used instead of 1.0 mol / L sodium acetate.

Example 6 (Preparation of Refolding Agent 6)
In Example 3, instead of 1.0 mol / L disodium hydrogen phosphate, 8.0 mol / L disodium hydrogen phosphate was used in the same manner as in Example 3, except that the refolding agent (R- 6) was prepared.

Example 7 (Preparation of Refolding Agent 7)
In Example 3, instead of 1.0 mol / L disodium hydrogen phosphate, 0.27 mol / L disodium hydrogen phosphate was used in the same manner as in Example 3, except that the refolding agent (R- 7) was prepared.

Example 8 (Preparation of Refolding Agent 8)
In Example 1, 1.0 mol / L sodium formate instead of 1.0 mol / L sodium acetate, 0.10 wt% sorbitan monolaurate instead of 0.10 wt% oleyl alcohol ethylene oxide 18 mol adduct A refolding agent (R-8) was prepared in the same manner as in Example 1 except that an EO adduct (“TWEEN 20” manufactured by Wako Pure Chemical Industries, Ltd.) was used.

Example 9 (Preparation 9 of refolding agent)
In Example 3, instead of 1.0 mol / L disodium hydrogen phosphate, 1.0 mol / L adenosine monophosphate sodium salt (“AMP”: manufactured by Wako Pure Chemical Industries), 0.10 wt% oleyl alcohol ethylene A refolding agent (R) was prepared in the same manner as in Example 3 except that 0.10 wt% lauryl alcohol EO11 mol adduct (“Emalmine NL-110” manufactured by Sanyo Chemical Industries) was used instead of the oxide 18 mol adduct. -9) was prepared.

Example 10 (Preparation of Refolding Agent 10)
The same procedure as in Example 1 was carried out except that, instead of the 0.10 wt% oleyl alcohol ethylene oxide 18 mol adduct, 1.33 wt% oleyl alcohol ethylene oxide 18 mol adduct was used. (R-10) was prepared.

Example 11 (Preparation of Refolding Agent 11)
The same procedure as in Example 1 was carried out except that 0.013 wt% oleyl alcohol ethylene oxide 18 mol adduct was used instead of 0.10 wt% oleyl alcohol ethylene oxide 18 mol adduct. (R-11) was prepared.

Example 12 (Preparation of Refolding Agent 12)
In Example 3, the same procedure as in Example 1 was used, except that 1.33% by weight of sorbitan monooleate EO adduct was used instead of 0.10% by weight of sorbitan monooleate EO adduct. -12) was prepared.

Example 13 Preparation of Refolding Agent 13
In Example 3, the same procedure as in Example 1 was carried out except that 0.013% by weight of sorbitan monooleate EO adduct was used instead of 0.10% by weight of sorbitan monooleate EO adduct. -13) was prepared.

Comparative Example 1 (Preparation 1 of comparative refolding agent)
To 20 mL sterilized vials, 1.0 mol / L sodium acetate and 50 mmol / L Tris buffer (pH = 8) were added respectively, dissolved in sterilized water, and refolding agent (R′− 1) was prepared.

Comparative Example 2 (Preparation 2 of comparative refolding agent)
To each 20 mL sterilized vial, 1.0 mol / L disodium hydrogen phosphate and 50 mmol / L Tris buffer (pH = 8) were added, respectively, dissolved in sterile water and refolding agent ( R′-2) was prepared.

Comparative Example 3 (Preparation 3 of comparative refolding agent)
In a 20 mL sterilized vial, 0.10 wt% oleyl alcohol ethylene oxide 18 mol adduct (“Emalmin 180” manufactured by Sanyo Chemical Industries), 50 mmol / L Tris buffer (pH = 8) And refolding agent (R′-3) was prepared by dissolving in sterilized water.

Comparative Example 4 (Preparation 4 of comparative refolding agent)
In a 20 mL sterilized vial, 1.0 mol / L sodium acetate, 0.10 wt% sodium lauryl sulfate (manufactured by Wako Pure Chemical Industries, Ltd.), 50 mmol / L Tris buffer (pH = 8) And refolding agent (R′-4) was prepared by dissolving in sterilized water.

Comparative Example 5 (Preparation 5 of comparative refolding agent)
In a 20 mL sterilized vial, 1.0 mol / L sodium acetate, 0.10 wt% coconut oil fatty acid amidopropyl betaine (“Levon 2000” manufactured by Sanyo Chemical Industries), 50 mmol / L Tris buffer ( Each was added so that pH = 8) and dissolved with sterilized water to prepare a refolding agent (R′-5).

Comparative Example 6 (Preparation 5 of comparative refolding agent)
In a 20 mL sterilized vial, 1.0 mol / L sodium acetate, 0.10 wt% cetyltrimethylammonium (manufactured by Wako Pure Chemical Industries, Ltd.), 50 mmol / L Tris buffer (pH = 8) And refolding agent (R′-6) was prepared by dissolving in sterilized water.

Example 14
To a 1.5 mL sterilized Eppendorf tube, add 1 mg of 2 mg of lysozyme (“Lysozyme Chloride” manufactured by Nacalai Tesque, hereinafter the same) and the unfolding solution prepared in Production Example 1 at 4 ° C. Leave it overnight to unfold the lysozyme.
0.75 mL of the refolding agent (R-1) prepared in Example 1 was added to 0.25 mL of this unfolded protein solution {concentration of compound (C) in the system = 0.75 mol / L; The concentration of compound (B) in the system = 0.075% by weight} was left at 4 ° C. overnight to perform refolding.

Example 15
In Example 14, refolding was performed in the same manner as in Example 14 except that (R-2) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 16
In Example 14, refolding was performed in the same manner as in Example 14 except that (R-3) was used instead of (R-1). (Concentration of compound (A) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 17
In Example 14, refolding was performed in the same manner as in Example 14 except that (R-4) was used instead of (R-1). (Concentration of compound (C) in the system = 6.0 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 18
In Example 14, refolding was performed in the same manner as in Example 14 except that (R-5) was used instead of (R-1). (Concentration of compound (C) in the system = 0.01 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 19
In Example 14, refolding was performed in the same manner as in Example 14 except that (R-6) was used instead of (R-1). (Concentration of compound (A) in the system = 6.0 mol / L, concentration of compound (B) in the system = 0.075% by weight)

Example 20
In Example 14, refolding was performed in the same manner as in Example 14 except that (R-7) was used instead of (R-1). (Concentration of compound (A) in the system = 0.2 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 21
In Example 14, refolding was performed in the same manner as in Example 14 except that (R-8) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 22
In Example 14, refolding was performed in the same manner as in Example 14 except that (R-9) was used instead of (R-1). (Concentration of compound (A) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 23
In Example 14, refolding was performed in the same manner as in Example 14 except that (R-10) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 1.0% by weight)

Example 24
In Example 14, refolding was performed in the same manner as in Example 14 except that (R-10) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0.01 wt%)

Example 25
In Example 14, refolding was performed in the same manner as in Example 14 except that (R-10) was used instead of (R-1). (Concentration of compound (A) in the system = 0.75 mol / L, concentration of compound (B) in the system = 1.0% by weight)

Example 26
In Example 14, refolding was performed in the same manner as in Example 14 except that (R-10) was used instead of (R-1). (Concentration of compound (A) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0.01 wt%)

Comparative Example 7
In Example 14, refolding was performed in the same manner as in Example 14 except that (R′-1) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0% by weight)

Comparative Example 8
In Example 14, refolding was performed in the same manner as in Example 14 except that (R′-2) was used instead of (R-1). (Concentration of compound (A) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0% by weight)

Comparative Example 9
In Example 14, refolding was performed in the same manner as in Example 14 except that (R′-3) was used instead of (R-1). (Concentration of compounds (A) and (C) in the system = 0 mol / L, concentration of compound (B) in the system = 0.075 wt%))

Comparative Example 10
In Example 14, refolding was performed in the same manner as in Example 14 except that (R′-4) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0% by weight)

Comparative Example 11
In Example 14, refolding was performed in the same manner as in Example 14 except that (R′-5) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0% by weight)

Comparative Example 12
In Example 14, refolding was performed in the same manner as in Example 14 except that (R′-6) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0% by weight)

<Measurement of lysozyme enzyme activity>
3 ml of a 0.5% by weight concentration Bacillus subtilis solution was placed in a 10 ml sterilized test tube, and the protein solution obtained in Examples 14 to 26 or Comparative Examples 7 to 12 was added with a 10 μl micropipette and mixed gently. Absorbance (A ₀ ) at 450 nm immediately after addition of protein was measured with an ultraviolet-visible spectrophotometer (manufactured by Shimadzu Corporation, UV-2550), and further A ₀ ) was measured, and the absorbance was measured again 5 minutes later. (A ₅ ) was measured, and the absorbance change DA = A ₀ -A ₅ for 5 minutes at 450 nm was calculated from these values.
Moreover, the blank aqueous solution which does not add an additive with the commercial item "lysozyme" of the same concentration as the lysozyme density | concentration of Examples 14-26 and Comparative Examples 7-12 was prepared, and 450 nm was used similarly to the above using this. The change in absorbance (DA _b ) for 5 minutes was calculated.

The enzyme activity after refolding was calculated using the following formula.
Enzyme activity (%) = (DA / DA _b ) × 100

  The enzyme activities of Examples 14 to 26 and Comparative Examples 7 to 12 are shown in Table 1.

According to Table 1, only the phosphorus-containing compound (A) of Comparative Example 8 or the carboxyl group-containing compound (C) of Comparative Example 7 alone cannot suppress lysozyme aggregation. Further, it can be seen from Table 1 that the nonionic surfactant (B) of Comparative Example 9 alone can suppress aggregation of lysozyme but cannot be used because lysozyme does not refold and does not exhibit activity. Furthermore, aggregation of lysozyme cannot be suppressed even in a combination of an anionic surfactant, an amphoteric surfactant and a cationic surfactant and a carboxyl group-containing compound instead of a nonionic surfactant as in Comparative Examples 10-12. Or activity is low.
On the other hand, it can be seen that the refolding agents of the present invention of Examples 14 to 26 combined with a nonionic surfactant do not cause aggregation of lysozyme as a protein and can be refolded with high efficiency.

Example 27
To a 1.5 mL sterilized Eppendorf tube, add 1 mg of 2 mg trypsin (manufactured by Nacalai Tesque: the same is used below) and the unfolding solution prepared in Production Example 1 and leave at 4 ° C. overnight. Unfolded the lysozyme.
0.75 mL of the refolding agent (R-1) prepared in Example 1 was added to 0.25 mL of this unfolded protein solution {concentration of compound (C) in the system = 0.75 mol / L; The concentration of compound (B) in the system = 0.075% by weight} was left at 4 ° C. overnight to perform refolding.

Example 28
In Example 27, refolding was performed in the same manner as in Example 27 except that (R-2) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 29
In Example 27, refolding was performed in the same manner as in Example 27 except that (R-3) was used instead of (R-1). (Concentration of compound (A) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 30
In Example 27, refolding was performed in the same manner as in Example 27 except that (R-4) was used instead of (R-1). (Concentration of compound (C) in the system = 6.0 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 31
In Example 27, refolding was performed in the same manner as in Example 27 except that (R-5) was used instead of (R-1). (Concentration of compound (C) in the system = 0.01 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 32
In Example 27, refolding was performed in the same manner as in Example 27 except that (R-6) was used instead of (R-1). (Concentration of compound (A) in the system = 6.0 mol / L, concentration of compound (B) in the system = 0.075% by weight)

Example 33
In Example 27, refolding was performed in the same manner as in Example 27 except that (R-7) was used instead of (R-1). (Concentration of compound (A) in the system = 0.2 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 34
In Example 27, refolding was performed in the same manner as in Example 27 except that (R-8) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 35
In Example 27, refolding was performed in the same manner as in Example 27 except that (R-9) was used instead of (R-1). (Concentration of compound (A) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 36
In Example 27, refolding was performed in the same manner as in Example 27 except that (R-10) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 1.0% by weight)

Example 37
In Example 27, refolding was performed in the same manner as in Example 27 except that (R-10) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0.01 wt%)

Example 38
In Example 27, refolding was performed in the same manner as in Example 27 except that (R-10) was used instead of (R-1). (Concentration of compound (A) in the system = 0.75 mol / L, concentration of compound (B) in the system = 1.0% by weight)

Example 39
In Example 27, refolding was performed in the same manner as in Example 27 except that (R-10) was used instead of (R-1). (Concentration of compound (A) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0.01 wt%)

Comparative Example 13
In Example 27, refolding was performed in the same manner as in Example 27 except that (R′-1) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0% by weight)

Comparative Example 14
In Example 27, refolding was performed in the same manner as in Example 27 except that (R′-2) was used instead of (R-1). (Concentration of compound (A) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0% by weight)

Comparative Example 15
In Example 27, refolding was performed in the same manner as in Example 27 except that (R′-3) was used instead of (R-1). (Concentration of compounds (A) and (C) in the system = 0 mol / L, concentration of compound (B) in the system = 0.075 wt%))

Comparative Example 16
In Example 27, refolding was performed in the same manner as in Example 27 except that (R′-4) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0% by weight)

Comparative Example 17
In Example 27, refolding was performed in the same manner as in Example 27 except that (R′-5) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0% by weight)

Comparative Example 18
In Example 27, refolding was performed in the same manner as in Example 27 except that (R′-6) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0% by weight)

<Measurement of trypsin enzyme activity>
3 ml of 0.35 mmol / L azocol (manufactured by Tokyo Chemical Industry) solution is placed in a 10 ml sterilized test tube, and the protein solution obtained in Examples 27 to 39 or Comparative Examples 13 to 18 is added with a 10 μl micropipette and lightly added. Stir. Absorbance (A ₀ ) at 540 nm immediately after adding the protein was measured with an ultraviolet-visible spectrophotometer (manufactured by Shimadzu Corporation, UV-2550), and further A ₀ was measured. A ₅ ) was measured, and the absorbance change DA = A ₅ -A ₀ for 5 minutes at 540 nm was calculated from these values.
Moreover, the blank aqueous solution which does not add an additive other than the commercial product "trypsin" of the same concentration as the trypsin density | concentration of Examples 27-39 and Comparative Examples 13-18 is prepared, and it is 450 nm similarly to the above using this. The change in absorbance (DA _b ) for 5 minutes was calculated.

The enzyme activity after refolding was calculated using the following formula.
Enzyme activity (%) = (DA / DA _b ) × 100

  The enzyme activities of Examples 27 to 39 and Comparative Examples 13 to 18 are shown in Table 2.

According to Table 2, only the phosphorus-containing compound (A) of Comparative Example 14 or the carboxyl group-containing compound (C) of Comparative Example 13 alone cannot suppress lysozyme aggregation. Table 1 shows that the nonionic surfactant (B) of Comparative Example 15 alone can suppress aggregation of lysozyme but cannot be used because lysozyme does not refold and does not exhibit activity. Furthermore, aggregation of lysozyme cannot be suppressed even with a combination of an anionic surfactant, an amphoteric surfactant and a cationic surfactant and a carboxyl group-containing compound instead of a nonionic surfactant as in Comparative Examples 16-18. Or activity is low.
On the other hand, it can be seen that the refolding agents of Examples 27 to 39 of the present invention combined with a nonionic surfactant do not cause aggregation of lysozyme as a protein and can be refolded with high efficiency.

Example 40
To a 1.5 mL sterilized Eppendorf tube, add 1 mg of 2 mg of β-lactoglobulin (manufactured by Wako Pure Chemical Industries, Ltd., hereinafter the same) and the unfolding solution prepared in Production Example 1, and add 1 mL at 4 ° C. It was allowed to stand overnight to unfold β-lactoglobulin.
0.75 mL of the refolding agent (R-1) prepared in Example 1 was added to 0.25 mL of this unfolded protein solution {concentration of compound (C) in the system = 0.75 mol / L; The concentration of compound (B) in the system = 0.075% by weight} was left at 4 ° C. overnight to perform refolding.

Example 41
In Example 40, refolding was performed in the same manner as in Example 40 except that (R-2) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 42
In Example 40, refolding was performed in the same manner as in Example 40 except that (R-3) was used instead of (R-1). (Concentration of compound (A) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 43
In Example 40, refolding was performed in the same manner as in Example 40 except that (R-4) was used instead of (R-1). (Concentration of compound (C) in the system = 6.0 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 44
In Example 40, refolding was performed in the same manner as in Example 40 except that (R-5) was used instead of (R-1). (Concentration of compound (C) in the system = 0.01 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 45
In Example 40, refolding was performed in the same manner as in Example 40 except that (R-6) was used instead of (R-1). (Concentration of compound (A) in the system = 6.0 mol / L, concentration of compound (B) in the system = 0.075% by weight)

Example 46
In Example 40, refolding was performed in the same manner as in Example 40 except that (R-7) was used instead of (R-1). (Concentration of compound (A) in the system = 0.2 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 47
In Example 40, refolding was performed in the same manner as in Example 40 except that (R-8) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 48
In Example 40, refolding was performed in the same manner as in Example 40 except that (R-9) was used instead of (R-1). (Concentration of compound (A) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0.075 wt%)

Example 49
In Example 40, refolding was performed in the same manner as in Example 40 except that (R-10) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 1.0% by weight)

Example 50
In Example 40, refolding was performed in the same manner as in Example 40 except that (R-10) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0.01 wt%)

Example 51
In Example 40, refolding was performed in the same manner as in Example 40 except that (R-10) was used instead of (R-1). (Concentration of compound (A) in the system = 0.75 mol / L, concentration of compound (B) in the system = 1.0% by weight)

Example 52
In Example 40, refolding was performed in the same manner as in Example 40 except that (R-10) was used instead of (R-1). (Concentration of compound (A) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0.01 wt%)

Comparative Example 19
In Example 40, refolding was performed in the same manner as in Example 40 except that (R′-1) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0% by weight)

Comparative Example 20
In Example 40, refolding was performed in the same manner as in Example 40 except that (R′-2) was used instead of (R-1). (Concentration of compound (A) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0% by weight)

Comparative Example 21
In Example 40, refolding was performed in the same manner as in Example 40 except that (R′-3) was used instead of (R-1). (Concentration of compounds (A) and (C) in the system = 0 mol / L, concentration of compound (B) in the system = 0.075 wt%))

Comparative Example 22
In Example 40, refolding was performed in the same manner as in Example 40 except that (R′-4) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0% by weight)

Comparative Example 23
In Example 40, refolding was performed in the same manner as in Example 40 except that (R′-5) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0% by weight)

Comparative Example 24
In Example 40, refolding was performed in the same manner as in Example 40 except that (R′-6) was used instead of (R-1). (Concentration of compound (C) in the system = 0.75 mol / L, concentration of compound (B) in the system = 0% by weight)

<Measurement of β-lactoglobulin refolding rate>
The protein solutions obtained in Examples 40 to 52 or Comparative Examples 19 to 24 were purified through a simple gel filtration column (“PD-10”: manufactured by GE Life Sciences) at 4 ° C.
Thereafter, the protein was separated by cation exchange chromatography (“AKTA Explorer”: manufactured by GE Life Sciences) at 4 ° C. Since two peaks appear (P1 is the peak of the first active βLG and P2 is the peak of the later modified βLG), each two peak areas {P1 and P2 peak areas (S _P1 , S _P2 ) } Was calculated.

The refolding rate was calculated using the following formula.
Enzyme activity (%) = {S _P1 / (S _P1 + S _P2 )} × 100

  Table 3 shows enzyme activities of Examples 40 to 52 and Comparative Examples 19 to 24.

According to Table 3, only the phosphorus-containing compound (A) of Comparative Example 20 and the carboxyl group-containing compound (C) of Comparative Example 19 alone cannot suppress lysozyme aggregation. Further, it can be seen from Table 1 that the nonionic surfactant (B) of Comparative Example 21 alone can suppress aggregation of lysozyme but cannot be used because lysozyme does not refold and does not exhibit activity. Furthermore, aggregation of lysozyme cannot be suppressed even with a combination of an anionic surfactant, an amphoteric surfactant and a cationic surfactant and a carboxyl group-containing compound instead of a nonionic surfactant as in Comparative Examples 22-24. Or activity is low.
On the other hand, it can be seen that the refolding agents of Examples 40 to 52 of the present invention combined with a nonionic surfactant do not cause aggregation of lysozyme as a protein and can be refolded with high efficiency.

By using the refolding agent and protein production method of the present invention, various useful proteins can be efficiently produced in high yield without aggregation.
Examples of the protein obtained by the protein production method of the present invention include enzymes and recombinant proteins.

Claims (12)

An unfolded protein refolding agent comprising a phosphorus-containing compound (A) having a group represented by the general formula (1) and a nonionic surfactant (B) as essential components (I ).

  The refolding agent according to claim 1, which is used at a concentration of 0.2 to 6 mol / L of the phosphorus-containing compound (A) in the system in the refolding step.   The refolding agent according to claim 1 or 2, wherein the phosphorus-containing compound (A) is at least one selected from the group consisting of inorganic phosphoric acid, alkyl phosphate ester, sugar phosphate ester and salts thereof.   The phosphorus-containing compound (A) is at least one selected from the group consisting of phosphoric acid, pyrophosphoric acid, polyphosphoric acid, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, and salts thereof. 4. The refolding agent according to any one of 3.   An unfolded protein refolding agent comprising an oxycarbonyl group-containing compound (C) having at least one group selected from the group consisting of a carboxyl group, a carboxylate anion group and an ester group, and a nonionic surfactant Refolding agent (II) containing agent (B) as an essential component.   The refolding agent according to claim 5, which is used at a concentration of 0.01 to 6 mol / L of the oxycarbonyl group-containing compound (C) in the system in the refolding step.   The refolding agent according to claim 5 or 6, wherein the oxycarbonyl group-containing compound (C) is a compound having at least one carboxyl group or carboxylate anion group in the molecule.   The refolding agent according to any one of claims 5 to 7, wherein the oxycarbonyl group-containing compound (C) is at least one selected from the group consisting of formic acid, acetic acid, propionic acid, lactic acid, tartaric acid and salts thereof.   The refolding agent according to any one of claims 1 to 8, wherein the nonionic surfactant (B) is a higher alcohol alkylene oxide adduct and / or an alkylene oxide adduct of a polyhydric alcohol.   The refolding agent according to any one of claims 1 to 9, which is used at a concentration of 0.01 to 1% by weight as a concentration of the nonionic surfactant (B) in the system in the refolding step.   The refolding agent according to any one of claims 1 to 10, wherein the protein is a protein containing an S-S bond in the molecule.   The manufacturing method of protein including the process of refolding protein with the refolding agent in any one of Claims 1-11.