JP2019210258A - Protein refolding agent, protein refolding method, and protein renaturation method - Google Patents

Abstract

To provide a protein refolding agent capable of efficiently refolding protein.SOLUTION: A protein refolding agent comprises as an active ingredient at least one selected from the group consisting of compounds represented by the formula (I) in the figure, and salts and solvates thereof. In the formula, X is a substituted or unsubstituted, linear or branched, divalent hydrocarbon group where the number of carbon atoms is from 1 to 10 inclusive, or a substituted or unsubstituted polyoxyalkylene group where the number of repeats is from 1 to 10 inclusive.SELECTED DRAWING: Figure 5

Description

  The present invention relates to a protein refolding agent, a protein refolding method, and a protein regeneration method.

  As interest in functional proteins, particularly antibody drugs, increases, the importance of methods for mass expression of specific proteins by genetic engineering techniques and methods for high yield recovery of target proteins are increasing. In particular, in the refolding process in which the target protein is recovered in an active state from the inclusion body of the protein produced during mass expression, the recovery yield may be greatly reduced. It is important in the field (Non-patent Document 1).

  In particular, proteins with disulfide bonds are equivalent to about 1/3 of the total protein, covering important and enormous factors for maintaining the homeostasis of the body, such as immunoglobulins and insulin, which are the basis of the immune system. A catalytic method for forming a disulfide bond has not yet been established. Particularly problematic is that many recombinant proteins aimed at protein preparations and the like form non-natural intermolecular and intramolecular disulfide bonds to form inactive substances. In the case of a protein having a plurality of disulfide bonds, the cross-linking mode of natural disulfide bonds, that is, the efficiency of the oxidative refolding process greatly depends on the yield.

  A typical control method is disulfide bond shuffling. It is a method that leads to the most stable natural structure by coexisting a reducing agent and an oxidizing agent in the refolding process and repeatedly forming and breaking disulfide bonds. Typical reducing agents and oxidizing agents used in this case are glutathione (GSH), β-mercaptoethanol (β-ME), dithiothreitol (DTT), and oxidized glutathione (GSSG), respectively. For example, in the case of RNase A, the recovery yields in the presence and absence (under air) of GSH / GSSG are compared, and in the presence of GSH / GSSG, the enzyme at the same time in the initial refolding process is compared. The activity is about twice as high and the speed of recovery of activity (1/2 activity recovery time) is about 2.8 times faster (Non-patent Document 2). Moreover, as a small molecule reducing agent (and an oxidizing agent combined therewith) used in oxidative refolding, GSH / GSSG (Non-patent Document 2), GSH + (±) -trans-1,2-bis (2-mercaptoacetamide) ) Cyclohexane (BMC) / GSSG (Non-patent Document 3), aromatic thiols and their disulfides (Non-patent Document 4), cyclic selenoxide (Non-patent Document 5), Cys A peptide having a -XX-Cys structure (X is an arbitrary amino acid and Cys is cysteine) (Non-Patent Document 6) and selenoglutathione (Non-Patent Document 7) are known. However, cyclic selenoxide and selenoglutathione have a problem of toxicity due to the use of heavy metal selenium. In addition, since BMC is used as an auxiliary agent for GSH, there is a problem in that it is necessary to add a large amount of additives compared to the GSH / GSSG system. Since aromatic thiols are substances having a benzene ring, there has been a problem in that non-specific adsorption due to hydrophobic interaction with proteins can occur. For peptides having a Cys-XX-Cys structure, structural instability such as degradation due to protease contamination and high cost for the synthesis were problems.

  Therefore, a refolding agent that can refold proteins with high efficiency while avoiding the problems of the prior art has been desired.

S. Yamaguchi et al. Biotechno. J. et al. , 2013, 8, 17-31 A. K. Ahmed et al. , J .; Biol. Chem. 1975, 250, 8477-8482. K. J. et al. Woycechosky et al. , Chem. Biol. 1999, 6, 871-879. D. J. et al. Madar et al. , J .; Biotech. , 2009, 142, 214-219 K. Arai, K et al. , Chem. Eur. J. et al. , 2011, 17, 481-485 W. J. et al. Lees et al. Curr. Opin. Chem. Biol. , 2008, 12, 740-745 J. et al. Belt et al. Biochemistry. 2007 May 8; 46 (18): 5382-90

  Therefore, an object of the present invention is to provide a protein refolding agent, a protein refolding method, and a protein regeneration method that give high refolding efficiency.

  As a result of various studies on means for solving the above-mentioned problems, the present inventors have achieved high refolding by using a compound represented by formula (I) having a specific structure, and salts and solvates thereof. We have found that efficiency can be achieved and have completed the present invention.

［式中、
Ｘは、置換若しくは非置換の直鎖状若しくは分岐鎖状の炭素数１以上１０以下の二価炭化水素基、又は置換若しくは非置換の繰り返し数１以上１０以下のポリオキシアルキレン基である］
で表される化合物、及びその塩及び溶媒和物からなる群から選択される少なくとも１種を有効成分として含む、タンパク質のリフォールディング剤。
［２］Ｘが、置換若しくは非置換の直鎖状若しくは分岐鎖状の炭素数１以上５以下の二価炭化水素基である、上記［１］に記載のリフォールディング剤。
［３］Ｘが、非置換であるか、又はヒドロキシル、カルボキシル及びアミノから選択される１種以上により置換されている、上記［１］に記載のリフォールディング剤。
［４］Ｘが、非置換であるか、又はヒドロキシル、カルボキシル及びアミノから選択される１種以上により置換されている、上記［２］に記載のリフォールディング剤。
［５］上記［１］〜［４］のいずれかに記載のリフォールディング剤の存在下で、アンフォールディングされたタンパク質を処理する工程を含む、タンパク質のリフォールディング方法。
［６］上記［１］〜［４］のいずれかに記載のリフォールディング剤の存在下で、アンフォールディングされたタンパク質を処理してリフォールディングする工程を含む、タンパク質の再生方法。 That is, the gist of the present invention is as follows.
[1] The following formula (I):

[Where:
X is a substituted or unsubstituted linear or branched divalent hydrocarbon group having 1 to 10 carbon atoms, or a substituted or unsubstituted polyoxyalkylene group having 1 to 10 repeating groups.
A protein refolding agent comprising, as an active ingredient, at least one selected from the group consisting of a compound represented by the formula:
[2] The refolding agent according to the above [1], wherein X is a substituted or unsubstituted linear or branched divalent hydrocarbon group having 1 to 5 carbon atoms.
[3] The refolding agent according to the above [1], wherein X is unsubstituted or substituted with one or more selected from hydroxyl, carboxyl and amino.
[4] The refolding agent according to the above [2], wherein X is unsubstituted or substituted with one or more selected from hydroxyl, carboxyl and amino.
[5] A protein refolding method comprising a step of treating an unfolded protein in the presence of the refolding agent according to any one of [1] to [4].
[6] A method for regenerating a protein comprising a step of treating and refolding an unfolded protein in the presence of the refolding agent according to any one of [1] to [4].

  The refolding agent according to the present invention can refold proteins with high efficiency.

FIG. 1A is a diagram showing the structure of bovine pancreatic trypsin inhibitor (BPTI) and the disulfide bond crosslinking position of each disulfide bond species in its folding pathway. FIG. 1B is a diagram showing the relationship between the HPLC retention time and each disulfide bond species when determining the disulfide bond crosslinking position of BPTI using reverse phase HPLC. FIG. 2A is a diagram showing the results of reverse-phase HPLC analysis when BPTI refolding reaction was performed using Example 1 (1 mM GdnSH / 0.2 mM GSSG). FIG. 2B is a diagram showing the results of reversed-phase HPLC analysis when BPTI refolding reaction was performed using Comparative Example 1 (1 mM GSH / 0.2 mM GSSG). FIG. 2C shows a reversed-phase HPLC analysis result when BPTI refolding reaction was performed using Comparative Example 2 (1 mM β-mercaptoethanol (β-ME) / 1 mM arginine (Arg) /0.2 mM GSSG). FIG. FIG. 2D shows the results of reversed-phase HPLC analysis in the case of performing the refolding reaction of BPTI using Comparative Example 3 (1 mM β-mercaptoethanol (β-ME) / 1 mM guanidine (Gdn) /0.2 mM GSSG). FIG. FIG. 3 is a graph showing the natural (N) BPTI yield of 60 minutes of BPTI refolding reaction time using Example 1 and Comparative Example 1-3. FIG. 4A is a diagram showing evaluation of introduction of a disulfide bond into reduced RNase A according to Example 2 (1 mM GdnSH / 0.2 mM GSSG). FIG. 4B is a diagram showing evaluation of introduction of a disulfide bond into reduced RNase A by Comparative Example 4 (1 mM GSH / 0.2 mM GSSG). FIG. 5 is a graph showing evaluation of activity recovery of reduced modified RNase A according to Example 3 (1 mM GdnSH / 0.2 mM GSSG) and Comparative Example 5 (1 mM GSH / 0.2 mM GSG).

［式中、
Ｘは、置換若しくは非置換の直鎖状若しくは分岐鎖状の炭素数１以上１０以下の二価炭化水素基、又は置換若しくは非置換の繰り返し数１以上１０以下のポリオキシアルキレン基である］で表される化合物、及びその塩及び溶媒和物からなる群から選択される少なくとも１種を有効成分として含むことを特徴とする（以下、本発明のリフォールディング剤ともいう）。本発明のリフォールディング剤によれば、高い効率でタンパク質をリフォールディングすることができる。以下、式（Ｉ）で表される化合物、及びその塩及び溶媒和物を単に式（Ｉ）で表される化合物ともいう。式（Ｉ）で表される化合物は、チオール基とグアニジン残基とを置換基Ｘを介して組み合せた構造を有する。チオール基を有する化合物（例えばβ−メルカプトエタノール）とグアニジンは、それぞれタンパク質内ジスルフィド結合の還元及び水素結合の切断に用いられる物質である。しかしながら、両構造を結合させた構造を有する化合物を用いることにより、それぞれを別々に用いた場合と比較して相乗的にリフォールディング効率を向上させることができることは知られていなかった。理論に拘束されるものではないが、式（Ｉ）で表される化合物は、塩基性でありかつ電子吸引性基であるグアニジン残基によりチオール基のｐＫａが最適化されているため、ジスルフィド結合のシャッフリングが効率的に起こり、特に酸化的リフォールディング過程に用いられる還元剤として良好に機能するものと考えられる。酸化的リフォールディング過程の高効率化は収率の向上にもつながるため、本発明のリフォールディング剤の使用は、機能性タンパク質等の開発にかかる時間短縮と収量向上によるコスト低下につながることも期待できる。また、式（Ｉ）で表される化合物は合成が比較的容易であるため低いコストで得られ、また特に酸性条件下及び／又は酸素非存在下で安定に保管することができる。また、式（Ｉ）で表される化合物は小分子であり、分子間力が小さいため一連のタンパク質のリフォールディング操作において取扱いが容易であり、特にリフォールディング反応後のタンパク質の単離・精製において、透析法やサイズ排除クロマトグラフィー法により、簡便に除去しやすいという利点を有する。 The present invention relates to a protein refolding agent, which is represented by the following formula (I):

[Where:
X is a substituted or unsubstituted linear or branched divalent hydrocarbon group having 1 to 10 carbon atoms, or a substituted or unsubstituted polyoxyalkylene group having 1 to 10 repeating groups. It contains at least one selected from the group consisting of the compound represented, and a salt and a solvate thereof as an active ingredient (hereinafter also referred to as a refolding agent of the present invention). According to the refolding agent of the present invention, a protein can be refolded with high efficiency. Hereinafter, the compound represented by the formula (I), and salts and solvates thereof are also simply referred to as a compound represented by the formula (I). The compound represented by the formula (I) has a structure in which a thiol group and a guanidine residue are combined through a substituent X. A compound having a thiol group (for example, β-mercaptoethanol) and guanidine are substances used for reduction of intraprotein disulfide bond and cleavage of hydrogen bond, respectively. However, it has not been known that by using a compound having a structure in which both structures are combined, the refolding efficiency can be synergistically improved as compared with the case where each is used separately. Without being bound by theory, the compound represented by the formula (I) has a disulfide bond because the pKa of the thiol group is optimized by the basic and electron-withdrawing group guanidine residue. Thus, it is considered that the shuffling occurs efficiently and particularly functions as a reducing agent used in the oxidative refolding process. Since the higher efficiency of the oxidative refolding process also leads to an increase in yield, the use of the refolding agent of the present invention is expected to lead to a reduction in cost due to a reduction in time required for the development of functional proteins and the like and an increase in yield. it can. Further, the compound represented by the formula (I) can be obtained at low cost because it is relatively easy to synthesize, and can be stably stored particularly under acidic conditions and / or in the absence of oxygen. In addition, the compound represented by the formula (I) is a small molecule, and since the intermolecular force is small, it is easy to handle in a series of protein refolding operations, particularly in the isolation and purification of the protein after the refolding reaction. The dialysis method and the size exclusion chromatography method have the advantage of being easily removed.

  For example, 1- (2-mercaptoethyl) guanidine (GdnSH), which is a compound represented by the formula (I), has a structure in which β-mercaptoethanol and guanidine are bonded. It has now been found that GdnSH can advance oxidative refolding of proteins at a higher speed and more efficiently than GSH that is generally used as a reducing agent used in the oxidative refolding process. Specifically, when the refolding of RNase A under the same conditions was compared with both GdnSH / GSSG or GSH / GSSG, the enzyme activity at the same time in the initial stage of refolding of GdnSH / GSSG It was shown to be about twice as high. In addition, when BPTI was refolded under the same conditions, the yield of the natural type obtained in the same time was approximately doubled when GdnSH was used compared with the case where β-mercaptoethanol and guanidine were simply mixed. Highly, it was suggested that a structure in which β-mercaptoethanol and guanidine are covalently bonded has a synergistic effect in promoting refolding.

  In the present specification, an “active ingredient” is a substance that acts on a protein in a certain state in the process of refolding the protein, and a substance that acts on the substance, and promotes protein refolding. It is not limited to the reducing agent in the oxidative refolding process as described above. As used herein, “protein refolding” includes (1) a step of unfolding a protein, (2) a step of refolding an unfolded protein, and (3) isolating the refolded protein. It is assumed to include the step of In the present specification, the term “refolding reaction” means the above step (2).

  The compound represented by the formula (I) is a protein unfolding agent (that is, a denaturing agent), an aggregation inhibitor, as an active ingredient in refolding for forming a disulfide bond, by appropriately adjusting use conditions such as concentration, It can function as a reducing agent, a structural modifier, a function auxiliary agent, and a function inhibitor, preferably a reducing agent. The compound represented by the formula (I) is a protein unfolding agent (ie, denaturing agent), agglutination as an active ingredient in refolding that does not require the formation of disulfide bonds by adjusting the conditions such as concentration as appropriate. It can function as an inhibitor, structure modifier, function aid, and function inhibitor.

  The “linear or branched divalent hydrocarbon group having 1 to 10 carbon atoms” in the above formula (I) may be either saturated or unsaturated, and may be linear or branched carbon. Examples thereof include an alkylene group having a number of 1 or more and 10 or less, and a linear or branched alkenylene group or an alkynylene group having a carbon number of 2 or more and 10 or less.

  As the alkylene group, one hydrogen atom at any position of the carbon skeleton of a linear or branched alkyl group having 1 to 10 carbon atoms is replaced with one additional bonding site, and a divalent moiety is substituted. What is formed is mentioned. The alkylene group is preferably linear from the viewpoint of optimizing the interaction between the thiol group and the guanidine residue. The alkylene group preferably has 1 to 5 carbon atoms, more preferably 1 to 3 carbon atoms. Examples of the linear or branched alkyl group having 1 to 5 carbon atoms include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl (sec-butyl) , Isobutyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, etc. Can be mentioned. Preferably, the linear or branched alkylene group having 1 to 5 carbon atoms is selected from the group consisting of methylene, ethylene, propylene, butylene and pentylene.

  As the alkenylene group, one hydrogen atom at any position of a straight or branched alkenyl carbon skeleton having 2 to 10 carbon atoms is replaced with one additional bonding site to form a divalent moiety. The main chain may have one or more double bonds. The alkenylene group is preferably linear from the viewpoint of optimizing the interaction between the thiol group and the guanidine residue. The alkenylene group preferably has 2 or more and 5 or less carbon atoms. Examples of the straight chain or branched chain alkenyl group having 2 to 5 carbon atoms are not limited thereto, but include ethenyl, 1-propenyl, 2-propenyl, 1-methylethenyl, 1-butenyl, 2-butenyl, 3 -Butenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl 1-methyl-1-butenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, -Methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-1-propene Le, 1,2-dimethyl-2-propenyl, 1-ethyl-1-propenyl, 1-ethyl-2-propenyl and position isomers thereof. Preferably, the straight chain or branched chain alkenylene group having 2 to 5 carbon atoms is ethenylene, 1-propenylene, 2-propenylene, 1-butenylene, 2-butenylene, 3-butenylene, 1- Selected from the group consisting of pentenylene, 2-pentenylene, 3-pentenylene and 4-pentenylene.

  As the alkynylene group, one hydrogen atom at any position of the carbon skeleton of a linear or branched alkynyl group having 2 to 10 carbon atoms is replaced with one additional bonding site, and And the main chain may have one or more triple bonds. The alkynylene group is preferably linear from the viewpoint of optimizing the interaction between the thiol group and the guanidine residue. The alkynylene group preferably has 2 or more and 5 or less carbon atoms. Examples of the linear or branched alkynyl group having 2 to 5 carbon atoms include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methyl-2-butynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl and 3- And methyl-1-butynyl. Preferably, the linear or branched alkynylene group having 2 to 5 carbon atoms is ethynylene, 1-propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 3-butynylene, 1-pentynylene, -Selected from the group consisting of pentynylene, 3-pentynylene and 4-pentynylene.

  The “polyoxyalkylene group” in the above formula (I) is a skeleton formed by repeating the same or different oxyalkylene units having 2 or more and 4 or less carbon atoms, specifically, polyoxyethylene group, polyoxypropylene group, etc. Is mentioned. The number of repeating oxyalkylene units of the polyoxyalkylene group is 1 or more and 8 or less, preferably 1 or more and 4 or less from the viewpoint of optimizing the interaction between the thiol group and the guanidine residue.

Specific examples of the “substituent” in the above formula (I) include amino, halogen (fluorine, chlorine, bromine or iodine), hydroxyl, oxo, nitro, CN, carboxyl, C ₁ -C ₆ -alkyl, C ₁ -C ₆ - _haloalkyl, C 1 -C ₆ - _alkoxy, C 1 -C ₆ - _haloalkoxy, C 3 -C ₆ - _cycloalkyl, C 3 -C ₆ - _cycloalkyl, C 2 -C ₆ - alkenyloxy C ₂ -C ₆ -alkynyloxy, C ₁ -C ₆ -alkoxycarbonyl, C ₁ -C ₆ -alkylcarbonyloxy, C ₆ -C ₁₅ -aryl (such as phenyl, naphthyl and anthryl), C ₇ -C ₁₈ - arylalkyl (benzyl, 1-phenethyl and _{2-phenethyl),} C 8 _{-C 18} - aryl alkenyl (styryl etc.) and From the viewpoint of suppressing adsorption to proteins, hydrophilic groups such as hydroxyl, carboxyl and amino are preferred. _Here, C n -C _m indicates the number of carbon atoms possible in the group. As used herein, the term “substituted” refers to replacing one or more atoms contained in X of formula (I) with the above substituents. Moreover, when substituting with hydrophobic groups, such as phenyl, in order to suppress adsorption | suction to protein, it is preferable that the number of the said substituent is 1-2.

  The compound represented by the above formula (I) can be synthesized by a known method (J. He et al., Nano Lett., 2008, 8, 2530-2534), for example, a compound having a disulfide bond. The amino groups at both ends can be converted to N, N′-bis (tert.) In the presence of a base (eg, DMAP, DIEA, pyridine, triethylamine, etc.) in an inert solvent (eg, methylene chloride, chloroform, DMF or a mixture thereof). -Butoxycarbonyl) -1H-pyrazole-1-carboxamidine was reacted with, and then subjected to acidic conditions such as hydrochloric acid to form a guanidine residue. Finally, dithiothreitol was used to convert the disulfide bond to a thiol group. Obtained by reduction.

  Any of the compounds represented by the above formula (I) may be in the form of a salt or a solvate. The salt is not particularly limited. For example, a salt with an alkali metal such as sodium or potassium; a salt with an alkaline earth metal such as magnesium or calcium; an ammonium salt; or hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, sulfurous acid, etc. Salt with inorganic acid; 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, tri And salts with organic acids such as fluoroacetic acid. Examples of the solvate include hydrates, alcohols (for example, methanol, ethanol, propanol, isopropanol), solvates with solvents such as acetone, tetrahydrofuran, dioxane, DMF, and DMSO.

  The refolding agent of the present invention contains, as an active ingredient, at least one selected from the group consisting of the compound represented by the formula (I), and salts and solvates thereof, and includes two or more arbitrarily selected compounds. In this case, the combination is not particularly limited. Even if the refolding agent of the present invention is sold or distributed in a state where the combination is mixed, it is sold or distributed in a separately packaged state as a kit or a set, etc. Also good. The compound represented by the above formula (I) is preferably packaged under acidic conditions (for example, about pH 2 to 3) and in the absence of oxygen (for example, ampoules) from the viewpoint of stability.

  Hereinafter, unless otherwise stated, embodiments of the refolding agent of the present invention comprising a compound represented by formula (I) as a reducing agent in the oxidative refolding of an unfolded protein will be described.

  One embodiment of the refolding agent of the present invention may further contain an oxidizing agent in addition to the compound represented by the above formula (I). Such an oxidizing agent is not particularly limited as long as it can be used together with the compound represented by the formula (I) as a reducing agent in oxidative refolding. For example, oxidized glutathione, oxidized dithiothreitol, seleno Glutathione oxidants, glutaredoxins, cystine, cystamine oxidants and selenocystamine oxidants can be mentioned, and glutathione oxidants are preferred because they are used in actual biological reactions. This embodiment preferably includes the compound represented by formula (I) and the oxidizing agent in a separately packaged form. Another embodiment of the refolding agent of the present invention contains the compound represented by formula (I) as a reducing agent in oxidative refolding, and is used for refolding proteins together with the above-mentioned oxidizing agent obtained separately. be able to.

  Another embodiment of the refolding agent of the present invention may contain other reducing agent in addition to the compound represented by the above formula (I) and optionally the above oxidizing agent. Examples of such a reducing agent include glutathione, β-mercaptoethanol, dithiothreitol, ascorbic acid, and cysteine. The combination of each component is not particularly limited, and is sold or distributed in a mixed state. However, it may be sold or distributed in the state of being individually packaged as a kit or a set. In the present embodiment, the content of the other reducing agent can be 10% by mass or less, preferably 5% by mass or less with respect to the compound represented by the formula (I).

  The target protein to be refolded using the refolding agent of the present invention is a peptide, polypeptide, protein, natural or artificial (chemical synthesis method, fermentation method, gene recombination method), etc. And complexes thereof. Regardless of the type of protein, for example, intracellular protein, extracellular protein, membrane protein, and nuclear protein are all included. Although not necessarily a protein having a disulfide bond, examples of suitable proteins include proteins having at least one disulfide bond. Specific examples include the following enzymes, recombinant proteins, and antibodies.

  Examples of the enzyme include a hydrolase, an isomerase, an oxidoreductase, a transferase, a synthetic enzyme, and a desorbing enzyme. Examples of hydrolases include protease, serine protease, amylase, lipase, cellulase, glucoamylase and the like. An example of the isomerase is glucose isomerase. Examples of the oxidoreductase include protein disulfide isomerase and peroxidase. Examples of the transferase include acyltransferase and sulfotransferase. Examples of the synthase include fatty acid synthase, phosphate synthase, citrate synthase and the like. Examples of the desorbing enzyme include pectin lyase.

  Examples of the recombinant protein include protein preparations and vaccines. Recombinant proteins produced by genetic engineering using prokaryotic organisms such as E. coli, eukaryotic organisms such as yeast, or heterologous expression systems such as cell-free extraction systems are often obtained as insoluble and inactive aggregates, so-called inclusion bodies. Therefore, the refolding agent of the present invention can be preferably used. Protein preparations include interferon α, interferon β, interleukin 1-12, growth hormone, erythropoietin, insulin, granular colony stimulating factor (G-CSF), tissue plasminogen activator (TPA), defensin, natriuresis Peptides, blood coagulation factor 2, somatomedin, glucagon, growth hormone releasing factor, serum albumin, calcitonin and the like can be mentioned. Examples of the vaccine include hepatitis A vaccine, hepatitis B vaccine, and hepatitis C vaccine. Although there is no limitation in particular as an antibody, A medical antibody is mentioned.

  In the present specification, the “unfolded protein” may be a protein unfolded by any method, but from the viewpoint of the refolding effect, it is unfolded by guanidine hydrochloride, urea, thiourea or a combination thereof. The protein is preferred. When the protein contains a disulfide bond in the molecule, in addition to the unfolding agent, β-mercaptoethanol, dithiothreitol, tris (2-carboxyethyl) phosphine (TCEP), thiophenol, etc. The protein may be unfolded by adding a reducing agent.

  Although the molecular weight of the unfolded protein is not particularly limited, a protein of about 1,000 to 10,000,000 is usually mentioned. From the viewpoint of the refolding effect, a protein having a molecular weight of 1,000 to 250,000 is preferred. According to the refolding agent of the present invention, since refolding can be performed with high efficiency, it can be applied to high molecular weight proteins.

  The present invention also relates to a protein refolding method including a step of treating an unfolded protein in the presence of the refolding agent of the present invention (hereinafter also referred to as the refolding method of the present invention). In the refolding method of the present invention, as a step of treating the unfolded protein in the presence of the refolding agent of the present invention (hereinafter, also simply referred to as a treatment step), the unfolded protein, the refolding agent, Specifically, there is a step of mixing the unfolded protein and the refolding agent in a refolding buffer and mixing them by stirring or the like, and further, if necessary, after the step. A step of standing for a certain period of time in order to proceed the refolding more fully is also included. Unless otherwise stated, the preferred embodiment of the refolding agent used in the refolding method of the present invention is referred to the above description of the refolding agent of the present invention.

  Although the processing time of the said process process is not restrict | limited especially as long as refolding is fully performed, For example, it is 5 minutes-24 hours, Preferably it is 10 minutes-3 hours. Moreover, temperature can be suitably selected according to the heat tolerance of the protein made into object, For example, it is the range of 0-100 degreeC, Preferably it is the range of 4-40 degreeC.

  The concentration of the compound represented by formula (I) (total amount) in the refolding agent used in the above treatment step is not particularly limited, but is usually 0 with respect to a solution containing the target protein (for example, a refolding buffer). 0.01 to 100 mM, preferably 0.05 to 10 mM, more preferably 0.1 to 5 mM. Moreover, the density | concentration of the unfolding protein contained in the solution (for example, refolding buffer solution) containing the said target protein is 0.01-100 micromol normally, Preferably it is 0.1-70 micromol, More preferably, it is 1-50 micromol.

  In the solution containing the target protein in the above treatment step (for example, refolding buffer), the molar ratio of the reducing agent (total amount) and the oxidizing agent (total amount), which are compounds represented by formula (I), is efficient. As long as the refolding proceeds, the number is not particularly limited. For example, the ratio is preferably 1: 1 to 20: 1, more preferably 2: 1 to 10: 1. Moreover, when an oxidizing agent is included in the refolding agent, the total amount with the oxidizing agent is included.

  The refolding buffer is not particularly limited as long as it does not have a concentration and composition that can cause loss of the function of the target protein. Specifically, amine-based buffers such as Tris buffer, MES buffer, and Tricine buffer are used. , Phosphate buffer, or various types of Good's buffer. The pH of the refolding buffer can be adjusted in the range of usually pH 4 to 10, preferably pH 5 to 9, more preferably pH 7 to 9.

  In addition to the refolding agent of the present invention, various additives can be added to the refolding buffer. 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, Examples include organic solvents such as ethanol and propanol. In addition to the refolding agent of the present invention and the above additives, a surfactant, a pH adjuster, or a protein stabilizer can be added to the buffer. A person skilled in the art can appropriately adjust the amount used.

  As the surfactant, any of nonionic surfactants, cationic surfactants, anionic surfactants and amphoteric surfactants can be used. Content in the case of using surfactant is 20 mass% or less normally with respect to the solution (for example, refolding buffer solution) containing target protein, Preferably it is 0.001-10 mass%, More preferably, 0.01- 5% by mass.

  Examples of the nonionic surfactant include adducts of higher alcohol alkylene oxide (hereinafter abbreviated as “AO”) (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 carbon alkyl, polypropylene glycol EO adduct and polyethylene glycol Examples include PO adducts, pluronic surfactants, fatty acid AO adducts, polyhydric alcohol type nonionic surfactants, and the like. Examples of the cationic surfactant include quaternary ammonium salt type cationic surfactants and amine salt type cationic cationic surfactants. Examples of the anionic surfactant include ether carboxylic acids or salts thereof, sulfate esters or ether sulfate esters and salts thereof, sulfonates, sulfosuccinates, fatty acids having a hydrocarbon group having 8 to 24 carbon atoms. Salts, acylated amino acid salts, and naturally occurring carboxylic acids and salts thereof (eg, chenodeoxycholic acid, cholic acid, deoxycholic acid, etc.). Examples of the amphoteric surfactant include betaine-type amphoteric surfactants and amino acid-type amphoteric surfactants.

  Examples of the pH adjuster include Tris (N-tris (hydroxymethyl) methylaminoethanesulfonic acid), HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), and a phosphate buffer. (For example, monosodium phosphate hydrogen 2 + aqueous hydrochloric acid solution, or monosodium dihydrogen phosphate + sodium hydroxide aqueous solution). The content in the case of using a pH adjuster is adjusted to be pH 4 to 10, preferably pH 5 to 9, more preferably pH 7 to 9, and a solution containing the target protein (for example, refolding buffer) On the other hand, it is usually 0.01 to 200 mM, preferably 0.05 to 150 mM, more preferably 0.1 to 100 mM.

  Examples of the protein stabilizer include reducing agents such as glutathione, β-mercaptoethanol, dithiothreitol, TCEP, ascorbic acid, and cysteine. The content in the case of using such a reducing agent is usually 0.01 to 100 mM, preferably 0.05 to 10 mM, more preferably 0.1 to 0.1% with respect to the solution containing the target protein (refolding buffer). 5 mM. When these reducing agents are contained in the refolding agent, the total amount with the contained reducing agent is used. Examples of the protein stabilizer include polyols, metal ions, chelating reagents and the like. Examples of polyols 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). Content in the case of using a protein stabilizer is 10 mass% or less normally with respect to the solution (refolding buffer solution) containing target protein, Preferably it is 0.001-10 mass%, More preferably, 0.01- 1% by mass.

  In addition, in the solution containing the target protein (refolding buffer), NDSB, arginine, glycine ethyl ester, glycinamide, arginine ethyl ester, arginine amide, etc. from the viewpoint of improving the balance between protein aggregation and refolding. Low molecular weight compounds may be included. In this case, the content is usually 0.01 to 100 mM, preferably 0.05 to 10 mM, more preferably 0.1 to 5 mM.

  In addition, the solution containing the target protein (refolding buffer) may contain an unfolding agent, that is, a denaturing agent (for example, guanidine hydrochloride or urea). In this case, the content of the unfolding agent is usually 0.01 to 100 mM, preferably 0.05 to 10 mM, more preferably 0.1 to 5 mM. The concentration may be adjusted by adding the refolding agent of the present invention to the unfolded protein suspension to dilute and lower the unfolding agent concentration, or the unfolded protein suspension Dialysis may be used to adjust the unfolding agent concentration by diluting and decreasing.

  The present invention also relates to a protein regeneration method comprising a step of treating and refolding an unfolded protein in the presence of the refolding agent of the present invention (hereinafter also referred to as the protein regeneration method of the present invention). ). The method for regenerating a protein of the present invention comprises a step of treating and refolding an unfolded protein in the presence of the refolding agent of the present invention (hereinafter also referred to as a refolding step), and further after the step. A step of isolating the refolded protein as necessary (hereinafter also referred to as an isolation step) is also included. Unless otherwise stated, the preferred embodiment of the protein regeneration method of the present invention is to cite the description of the refolding agent of the present invention and the refolding method of the present invention. Unless otherwise specified, the preferred embodiment of the refolding step is cited from the description of the processing step of the refolding method of the present invention.

  Examples of the isolation step include isolation of the target normal protein (refolded protein) from the protein suspension obtained in the refolding step using column chromatography or the like. Examples of the filler used for column chromatography include silica, dextran, agarose, cellulose, acrylamide, vinyl polymer and the like. Examples of commercially available products include Sephadex series, Sephacryl series, Sepharose series (hereinafter, Pharmacia), Bio-Gel series (Bio-Rad), and the like. Moreover, you may isolate the target normal protein (refolding protein) by the dialysis method.

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

(1) Synthesis of 1- (2-mercaptoethyl) guanidine (GdnSH) Synthesized according to the following scheme.

(1-1) Synthesis of Compound 2 N, N′-bis (tert-butoxycarbonyl) − with respect to a dehydrated DMF (Kanto Chemical, 15 mL) solution of Compound 1 (Nacalai, 0.340 g, 1.51 mmol) under nitrogen. 1H-pyrazole-1-carboxamidine (TCI, 0.993 g, 3.20 mmol) and triethylamine (sigma, 0.50 mL) were added, and the mixture was stirred at room temperature for 2 hours. Methylene chloride (AGC Chemical Company, 30 mL) was added, and washed with water (80 mL, 3 times) and saturated brine (50 mL, 1 time). Dehydrated sodium sulfate (Kishida Chemical) was added to the recovered methylene chloride solution, filtered, and the solvent was distilled off under reduced pressure. Compound 2 (0.397 g, yield 41%) was obtained by silica gel column chromatography (neutral silica gel, Kanto Chemical, developing solvent: ethyl acetate / hexane = 1/4 to 1/1 gradient).
¹ H NMR (CDCl ₃ , 25 ° C): 3.77 (4H, td, J = 6.4 and 5.5 Hz), 2.88 (4H, t, J = 6.4 Hz), 1.54 (24 H, s), 1.50 (12H, s ) ppm.

(1-2) Synthesis of Compound 3 Hydrochloric acid (1 mol / L, Kishida Kagaku, 20 mL) was added to a solution of Compound 2 (0.264 g, 0.415 mmol) in methanol (Gordo, 6 mL) under air at 30 ° C. Stir for 3 hours. The white solid obtained by evaporating the solvent under reduced pressure was dissolved in water (10 mL) and washed three times with methylene chloride (AGC Chemical Company, 10 mL). The collected aqueous solution was distilled off under reduced pressure to obtain Compound 3 (0.108 g, yield 95%).
¹ H NMR (D ₂ O, 25 ° C.): 3.44 (4H, t, J = 6.4 Hz), 2.81 (4H, t, J = 6.4 Hz) ppm.
¹³ C NMR (D ₂ O, 25 ° C.): 156.90, 39.78, 36.03 ppm.
ESI-TOF MS (0.1% formic acid mixed methanol): 237.0953 (theoretical mass of compound 3 + H ⁺ 237.0956).

(1-3) Synthesis of GdnSH Under nitrogen, compound 3 (0.105 g, 0.385 mmol) was dissolved in water (10 mL), and nitrogen bubbling was performed for 10 minutes. Dithiothreitol (nacalai, 0.118 g, 0.764 mmol) was added and stirred at 30 ° C. for 20 hours. Water (10 mL) was added, and the mixture was washed with chloroform (Kishida Chemical, 10 mL, 4 times) and a mixed solvent of 2-propanol and chloroform (10/70, both of which were Xida Chemical, 4 times). The recovered aqueous solution was evaporated under reduced pressure to obtain GdnSH (0.0882 g, yield 83%) as a colorless oil. From the thiol group determination method using 5,5′-dithiobis (2-nitrobenzoic acid) (nacalai), it was confirmed that 98 mol% of GdnSH was contained.
¹ H NMR (D ₂ O, 25 ° C.): 3.27 (2H, t, J = 6.4 Hz), 2.6 (2H, t, J = 6.4 Hz) ppm.
ESI-TOF MS (methanol): 120.0594 (theoretical mass of GdnSH + H ⁺ 120.0595).

(2) Preparation of reduced denatured protein Bovine Pancreas Trypsin Inhibitor (BPTI) (see References 1 and 2 below) and RNase A (see Reference Reference 3 below) were adopted as model substrates. . These are proteins having 3 and 4 disulfide bonds, respectively, and are generally used as model substrates in this field.
In order to prepare a reduced modified form of BPTI (Takara Bio) (reduced form of three disulfide bonds), 10 mg of BPTI was incubated in the presence of 8 M urea and 20 mM DTT at pH 8.0, 50 ° C. for 3 hours, Purified by HPLC. After confirming that all disulfide bonds of the purified sample were cleaved by MALDI-TOF / MS, the product was lyophilized and stored at −80 ° C. (see Reference Document 1 below).
In order to prepare a reduced modified form of RNase A (sigma) (reduced form of four disulfide bonds), 8 mg of RNase A was incubated in the presence of 6M guanidine hydrochloride and 100 mM DTT at pH 8.7, 25 ° C. for 2 hours, and 10 mM. Dialyzed against HCl. Further, dialysis was performed twice, and the solution was exchanged with 10 mM HCl except for DTT and the like (see Reference Document 3 below).
Reference 1: M.M. Okumura et al. J Biol Chem. 2014 289 (39): 27004-27018
Reference 2: J. S. Weissman et al. Science 1991 253 (5026): 1386-93
Reference 3: M.M. M.M. Lyles et al. Biochemistry. 1991 30 (3): 613-9

(3) Evaluation of ability to introduce disulfide bond into reduced modified BPTI In a buffer solution (50 mM Tris-HCl pH 7.5, 300 mM NaCl), the following reducing agent and the following reducing agent were added to 30 μM of the reduced modified BPTI prepared in (2) above. Oxidizing agent combination:
Example 1: 1 mM GdnSH / 0.2 mM GSSG (nakalai)
Comparative Example 1: 1 mM GSH (nakalai) /0.2 mM GSSG
Comparative Example 2: 1 mM β-mercaptoethanol (β-ME) (nakalai) / 1 mM arginine (Arg) (nakalai) /0.2 mM GSSG
Comparative Example 3: 1 mM β-mercaptoethanol (β-ME) (nakarai) / 1 mM guanidine (Gdn) (wako) /0.2 mM GSSG
Was incubated at 30 ° C. to perform a refolding reaction. The reaction solution was collected over time, and an equal amount of 1N HCl was added to quench the thiol group and disulfide bond exchange reaction. In order to identify the molecular species (see FIG. 1A) in the reaction solution, it was subjected to reverse phase HPLC. As shown in FIGS. 1A and B, the analysis using reverse phase HPLC can separate and identify each disulfide bond species when BPTI refolds, and can track the position of disulfide bond cross-linking over time. Note that the BPTI refolding path of FIG.
The measurement and analysis conditions for reverse phase HPLC are as follows:
The column was TSKgel Protein C4-300 column (4.6 × 150 mm; Tosoh Bioscience), and detected at an absorbance of 229 nm. Regarding mixing of an aqueous solution containing 0.05% trifluoroacetic acid (liquid A) and an acetonitrile solution containing 0.05% trifluoroacetic acid (liquid B), the volume increase rate of the ratio of liquid B in the mixed liquid is 0 to 15 Development was performed with a linear concentration gradient of 1% / min up to 1 min and 0.5% / min up to 15-115 min.

2A to D, when Comparative Example 1 (GSH / GSSG), Comparative Example 2 (β-mercaptoethanol / arginine / GSSG), and Comparative Example 3 (β-mercaptoethanol / guanidine / GSSG) were used, Even in the folding reaction time of 5 minutes, R (see FIG. 1A), which is a reductive modified form of BPTI (all thiol groups) remains, whereas in Example 1 (GdnSH / GSSG), almost all disappeared. It can be seen that the ability to introduce disulfide bonds was accelerated. 2A to 2D, BPTI of natural type (N) (see FIG. 1A) was found to be Comparative Example 1 (GSH / GSSG), Comparative Example 2 (β-mercaptoethanol / arginine / GSSG), and Comparative Example 3. When (β-mercaptoethanol / guanidine / GSSG) was used, it was formed only after the refolding reaction time was 30 minutes, whereas in Example 1 (GdnSH / GSSG), it was formed after 10 minutes. . In general, the formation of the natural form (N) refers to the rate of the exchange reaction between the thiol group and the disulfide bond once formed (the exchange reaction of N ′ and N ^* in the refolding pathway of FIG. 1A). This means that this exchange reaction has been accelerated.

  FIG. 3 shows a comparison of yields obtained by reverse phase HPLC with a refolding reaction time of 60 minutes. From FIG. 3, the yield of Example 1 using GdnSH is 51.3%, Comparative Example 1 using GSH (24.2%), and Comparative Example 2 using β-ME / Arg (25.8). %), And the yield was approximately twice that of Comparative Example 3 (27.6%) using β-ME / Gdn.

  From the above, Example 1 using GdnSH having a structure in which β-mercaptoethanol and guanidine are combined has a remarkable reaction rate and yield as compared with Comparative Example 3 in which each was separately used at the same concentration. It was shown to be high.

(4) Evaluation of ability to introduce disulfide bond into reduction-modified RNase A Next, RNase A was employed for the purpose of expanding general-purpose examples. In order to observe the process of forming a disulfide bond in the RNase A molecule, 4-acetamido-4′-maleimidyl stilbene-2,2′-disulfonic acid (AMS, Invitrogen), which is a free thiol group modifying reagent, was used. Using, only the thiol group not forming a disulfide bond was selectively modified and separated by polyacrylamide gel electrophoresis (SDS-PAGE). As the conditions for the refolding reaction, the following combination of reducing agent and oxidizing agent was applied to 8 μM of the reduced modified RNase A prepared in the above (2) in a buffer solution (50 mM Tris-HCl pH 7.5, 300 mM NaCl):
Example 2: 1 mM GdnSH / 0.2 mM GSSG
Comparative Example 4: 1 mM GSH / 0.2 mM GSSG
Incubated at 30 ° C. in the presence of The reaction solution was collected over time, and the reaction was quenched by adding a final concentration of 5 mM AMS. The degree of introduction of disulfide bonds in RNase A was evaluated by subjecting to electrophoresis (FIGS. 4A and B).
The electrophoresis measurement and analysis conditions are as follows:
A preparative sample was applied to a 14% polyacrylamide WIDE RANGE gel (nakalai), and electrophoresed and separated at 200 V for about 60 minutes. The protein was fixed with fixative (10% acetic acid, 50% methanol) and the gel was stained with Coomassie Brilliant Blue. The band intensity of the stained oxidized RNase A was quantified with analysis software Image Lab (Bio-Rad).
As a result, in the case of Comparative Example 4 (1 mM GSH / 0.2 mM GSSG), oxidized RNase A was not produced until the refolding reaction time was 60 minutes (FIG. 4B), but Example 2 (1 mM GdnSH / 0.2 mM GSSG). ) In the presence, an oxidized form was formed at a refolding reaction time of 10 minutes (FIG. 4A), and it was found that disulfide bond formation was promoted by GdnSH.

(5) Activity evaluation RNase A was adopted to evaluate the activity value after the refolding reaction. Combinations of the following reducing and oxidizing agents:
Example 3: 1 mM GdnSH / 0.2 mM GSSG
Comparative Example 5: 1 mM GSH / 0.2 mM GSG
A buffer solution (50 mM Tris-HCl pH 7.5, 300 mM NaCl) containing 8 μM RNase A was incubated at 30 ° C. to perform a refolding reaction. The reaction solution was collected over time and diluted with the same buffer containing adenosine 2 ′, 3′-cyclic monophosphate (cCMP) as a substrate for RNase A, and the final concentration was 2 μM RNase A, 0.4 mM cCMP. did. The change in absorbance at 284 nm was measured with a spectrophotometer (device name: HITACHI U-3900), and the nucleolytic activity of the refolded RNase A was quantified. The result is shown in FIG. FIG. 5 shows that the activity of RNase A is about 2.4 times faster in the presence of Example 3 (1 mM GdnSH / 0.2 mM GSSG) than in the case of Comparative Example 5 (1 mM GSH / 0.2 mM GSSG). It can be seen that has recovered.

  From the above, it can be seen that the protein can be refolded with high refolding efficiency by using the refolding agent of the present invention containing the compound represented by the formula (I) having a specific structure.

  The refolding agent of the present invention can be preferably applied to the refolding of a modified protein expressed from a recombinant gene, particularly the refolding of a large protein such as an antibody having many disulfide bonds.

Claims (6)

The following formula (I):

[Where:
X is a substituted or unsubstituted linear or branched divalent hydrocarbon group having 1 to 10 carbon atoms, or a substituted or unsubstituted polyoxyalkylene group having 1 to 10 repeating groups.
A protein refolding agent comprising, as an active ingredient, at least one selected from the group consisting of a compound represented by the formula:   The refolding agent according to claim 1, wherein X is a substituted or unsubstituted linear or branched divalent hydrocarbon group having 1 to 5 carbon atoms.   The refolding agent according to claim 1, wherein X is unsubstituted or substituted by one or more selected from hydroxyl, carboxyl and amino.   The refolding agent according to claim 2, wherein X is unsubstituted or substituted by one or more selected from hydroxyl, carboxyl and amino.   A method for refolding a protein, comprising a step of treating the unfolded protein in the presence of the refolding agent according to any one of claims 1 to 4.   A method for regenerating a protein, comprising a step of treating and refolding an unfolded protein in the presence of the refolding agent according to any one of claims 1 to 4.

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