Protein folding agent, protein folding composition, protein folding kit, and protein folding method

Abstract

Provided is a protein folding agent containing a compound (A) represented by general formula (A). In general formula (A), R1 to R6 are each independently a C1-24 aliphatic hydrocarbon group. Y1and Y2each independently represent a C1-6 aliphatic hydrocarbon group. Moreover, n is an integer of 1 or greater, and Xn- is an n-valent anion. Also provided is a protein folding composition containing the compound (A) and a protein aggregation inhibitor. Furthermore, also provided is a protein folding method including a step (a) for incubating a protein in the presence of the protein folding agent.

Description

Protein folding agent, protein folding composition, protein folding kit, and protein folding method

[0001] The present invention relates to a protein folding agent, a protein folding composition, a protein folding kit, and a protein folding method. This application claims priority under Japanese Patent Application No. 2024-191797, filed in Japan on October 31, 2024, the contents of which are incorporated herein by reference.

[0002] Protein folding is the reaction that refolds a denatured protein back into its active, native structure. Because denatured proteins have a tendency to aggregate irreversibly, the efficiency of protein folding is usually low. Compounds that promote protein folding are important additives in the production of industrially useful proteins, such as pharmaceutical proteins, as they allow for the efficient acquisition of active proteins.

[0003] Proteins can be broadly classified into two types: those that have disulfide bonds within their molecule and those that do not. In living organisms, folding proceeds through different mechanisms for each type. Folding of proteins without disulfide bonds is facilitated by molecular chaperones, such as GroEL. Folding of proteins with disulfide bonds is facilitated by oxidoreductases, such as Protein Disulfide Isomerase (PDI), which cause the formation and exchange of disulfide bonds. Proteins without disulfide bonds are often found inside cells, i.e., in the cytoplasm. On the other hand, many membrane proteins and proteins secreted extracellularly have disulfide bonds. Many proteins useful as pharmaceuticals, such as antibodies and insulin, also have disulfide bonds.

[0004] Compounds that promote the folding of proteins containing disulfide bonds have been reported, including glutathione (GSH / GSSH) (Non-Patent Document 1), nitrogen atom-containing thiol compounds (Patent Document 1), and thiol compounds having a cyclodextrin skeleton (Patent Document 2).

[0005] Protein folding, the process of refolding denatured proteins back to their native structure, is considered the final step in protein synthesis. Denatured proteins have a tendency to associate intermolecularly and are particularly prone to irreversible aggregation at high concentrations. Therefore, to prevent aggregation, protein folding is usually performed at dilute concentrations (around a few μM). The limitation of using dilute proteins in the folding reaction is one factor that reduces the efficiency of protein folding.

[0006] Japanese Patent Publication No. 2022-135954 Japanese Patent Publication No. 2023-127958

[0007] AK Ahmed, SW Schaffer, DB Wetlaufer, Nonenzymic reactivation of reduced bovine pancreatic ribonuclease by air oxidation and by glutathione oxidoreduction buffers. Journal of Biological Chemistry, Volume 250, Issue 21, 10 November 1975, Pages 8477-8482

[0008] Conventional protein folding agents used for folding proteins containing disulfide bonds are used at protein concentrations of several μM to tens of μM. No protein folding agents usable at protein concentrations of several hundred μM (e.g., 200 μM) or higher have been reported. If oxidative protein folding involving disulfide bond formation could be performed at protein concentrations of several hundred μM or higher, protein production efficiency could be dramatically increased.

[0009] Therefore, an object of the present invention is to provide a protein folding agent capable of promoting protein folding at a protein concentration of several hundred μM or more, a protein folding composition containing the protein folding agent, a protein folding kit, and a protein folding method using the protein folding agent.

[0010] The present invention includes the following aspects. [1] A protein folding agent containing a compound (A) represented by the following general formula (A).

[0011] [In the formula, R , ｎ－ ,

[0015] , １ , , ,

[0014] , ４ , ,

[0012] , １ ,

[0016] , ６ , <# ｎ－ , , ,

[0013] , , ｎ－ ~R ６ each independently represents an aliphatic hydrocarbon group having 1 to 24 carbon atoms. Y １ and Y <# ２ each independently represents an aliphatic hydrocarbon group having 1 to 10 carbon atoms which may contain an oxygen atom. n is an integer of 1 or more, and X <# ｎ－ represents an n-valent anion. ]

[0012] [2] The protein folding agent according to [1], wherein the compound (A) is a compound represented by the following general formula (A1).

[0013] [In the formula, R １ ~R ６ each independently represents an aliphatic hydrocarbon group having 1 to 24 carbon atoms. n is an integer of 1 or more, and X ｎ－ represents an n-valent anion. ]

[0014] [3] The protein folding agent according to [2], wherein the compound (A) is a compound represented by the following general formula (A1-1).

[0015] [In the formula, R １ and R ４ each independently represents an aliphatic hydrocarbon group having 1 to 24 carbon atoms. n is an integer of 1 or more, and X ｎ－ represents an n-valent anion. ]

[0016] [4] The protein folding agent according to [3], wherein the compound (A) is a compound represented by the following general formula (A1-1-1).

[0017] [In the formula, m1 and m2 each independently represent integers from 1 to 24. n is an integer greater than or equal to 1, and X ｎ－ This represents an n-valent anion.

[0018] [5] The X ｎ－ A protein folding agent according to any one of [1] to [4], wherein is a halide ion. [6] A protein folding composition comprising a protein folding agent according to any one of [1] to [5] and a protein aggregation inhibitor. [7] A protein folding composition according to [6], wherein the protein aggregation inhibitor is a surfactant. [8] A protein folding kit comprising a protein folding agent according to any one of [1] to [5]. [9] A protein folding kit according to [8], further comprising a protein aggregation inhibitor.

[10] A protein folding kit according to [8] or [9], further comprising a polar organic solvent.

[11] A method for folding a protein, comprising step (a) of incubating a protein in the presence of a protein folding agent according to any one of [1] to [5].

[12] A method for folding a protein according to

[11] , wherein step (a) is a step of incubating a protein in the presence of the protein folding agent and the protein aggregation inhibitor.

[13] A method for folding a protein according to

[11] or

[12] , further comprising the step (b) of adding a polar organic solvent to the folding reaction solution after step (a).

[14] A method for folding a protein according to any one of

[11] to

[13] , wherein the protein is an unfolding protein or a misfolding protein.

[0019] The present invention provides a protein folding agent capable of promoting protein folding at protein concentrations of several hundred μM or more, a protein folding composition containing the protein folding agent, a protein folding kit, and a protein folding method using the protein folding agent.

[0020] The structure of the compound (MeN-SS) synthesized in the examples was confirmed. １ The results of the 1H NMR measurement are shown. The results of the ESI-TOF mass spectrometry measurement confirming the structure of the compound (MeN-SS) synthesized in the example are shown. The structure of the compound (C16N-SS) synthesized in the example was confirmed. １ The results of the 1H NMR measurement are shown. The structure of the compound (C16N-SS) synthesized in the example was confirmed. １３ The results of the 13C NMR measurement are shown. The results of the MALDI-TOF mass spectrometry measurement confirming the structure of the compound (C16N-SS) synthesized in the example are shown. The results of evaluating the protein aggregation inhibitory effect of various ammonium lipids are shown. Reductively denatured Bovine Pancreatic Trypsin Inhibitor (BPTI) was used as the model protein. The results of the folding test of reductively denatured BPTI using CTAB are shown. The results of the folding test of reductively denatured BPTI using CTAB and the compound (MeN-SS) are shown. The results of the folding test of reductively denatured BPTI using CTAB and the compound (C16N-SS) are shown. The results of the test evaluating the effect of CTAB concentration on the folding of reductively denatured BPTI using CTAB and the compound (C16N-SS) are shown. This paper presents the results of a study evaluating the effect of acetonitrile added after incubation in the folding of reductively denatured BPTI using CTAB and compound (C16N-SS). It also shows the results of an evaluation of the protein aggregation inhibitory effect of compound (C16N-SS). Reductively denatured BPTI was used as the model protein. The structure of compound (C8N-SS) synthesized in the examples was confirmed. １The results of the 1H NMR measurement are shown. The structure of the compound (C8N-SS) synthesized in the example was confirmed. １３ The results of the 13C NMR measurement are shown. The results of the MALDI-TOF mass spectrometry measurement confirming the structure of the compound (C8N-SS) synthesized in the example are shown. The results of the MALDI-TOF mass spectrometry measurement confirming the structure of the compound (C8N-SS) synthesized in the example are shown. The results of the folding test of reductively modified BPTI using CTAB and the compound (C8N-SS) are shown. The results of the comparison over time of the formation rate of natural structure BPTI from the folding test of reductively modified BPTI using CTAB and each compound are shown. The results of the folding test of reductively modified hirudin using CTAB and the compound (C16N-SS) are shown. The results of the folding test of reductively modified hirudin using CTAB and the compound (C8N-SS) are shown. The results of the folding test of reductively modified hirudin using CTAB and the compound (MeN-SS) are shown. The results of the folding test of reductively modified hirudin using the compound (MeN-SS) are shown. The results of folding tests of reductively modified hirudin using GSSG are shown. The results of the folding tests of reductively modified hirudin using each compound are shown, comparing the formation rate of the natural structure hirudin over time. The results of folding tests of reductively modified endothelin-1 (ET-1) using CTAB and compound (C16N-SS) are shown. The results of the folding test of reductively modified ET-1 using GSSG are shown. The results of the folding tests of reductively modified ET-1 using each compound are shown, comparing the formation rate of the natural structure ET-1 over time. The results of the evaluation of the protein aggregation inhibitory effect of compound (C16N-SS) are shown. Reductively modified ET-1 was used as a model protein.

[0021] The term "comprise" means that components other than the component being discussed may be included. The term "consist of" means that components other than the component being discussed are not included. The term "consistently of" means that components other than the component being discussed are not included in a manner that performs a special function (such as a manner that completely negates the effect of the invention). In this specification, when "comprise" is used, it includes the "consist of" and "consistently of" manners.

[0022] Unless otherwise specified, "a," "an," and "the" encompass both singular and plural forms and are understood to mean "one or more."

[0023] A numerical range expressed using "~" means a range that includes the numbers written before and after "~" as the lower and upper limits, respectively.

[0024] If multiple upper and lower limits are specified for a particular parameter, any combination of these upper and lower limits can be used to create a suitable numerical range.

[0025] [Protein Folding Agent] A first aspect of this disclosure is a protein folding agent. The protein folding agent according to this aspect includes a compound represented by the following general formula (A) (hereinafter also referred to as "compound (A)").

[0026] [In the formula, R １ ~R ６ Each of these independently represents an aliphatic hydrocarbon group having 1 to 24 carbon atoms. １ and Y ２ Each of these independently represents an aliphatic hydrocarbon group having 1 to 10 carbon atoms, which may contain an oxygen atom. n is an integer greater than or equal to 1, and X ｎ－ This represents an n-valent anion.

[0027] <Compound (A)> Compound (A) is a compound represented by the general formula (A) above. In formula (A), R １ ~R ６ The number of carbon atoms in the aliphatic hydrocarbon group in is preferably 22 or less, more preferably 20 or less, even more preferably 18 or less, and even more preferably 16 or less, from the viewpoint of suppressing aggregation of compound (A) in the folding reaction solution. １ ~R ６ Examples of the range of carbon atoms in the aliphatic hydrocarbon group in R include 1-22, 1-20, 1-18, 1-16, etc. １ ~R ６ The number of carbon atoms in the aliphatic hydrocarbon group may be selected according to the hydrophobicity of the protein to be folded (hereinafter also referred to as the "target protein").

[0028] From the perspective of promoting the folding of the target protein, R １ and R ４ It is preferable that the number of carbon atoms is 6 or more, 8 or more, 10 or more, 12 or more, or 14 or more. １ and R ４ Examples of carbon atom numbers include 1-24, 6-24, 8-24, 10-24, 12-24, 14-24, 16-24, 1-20, 6-20, 8-20, 10-20, 12-20, 14-20, 16-20, 1-18, 6-18, 8-18, 10-18, 12-18, 14-18, 16-18, 1-16, 6-16, 8-16, 10-16, 12-16, 14-16, etc. ２ , R ３ , R ５ , and R ６ It is preferable that the number of carbon atoms is 10 or less, 8 or less, 6 or less, 4 or less, or 2 or less. ２ , R ３ , R ５ , and R ６ Examples of the number of carbon atoms include 1 to 10, 1 to 8, 1 to 6, 1 to 4, and 1 to 2. In some embodiments, R ２ , R ３ , R ５ , and R ６ The number of carbon atoms is 1 or 2.

[0029] From the viewpoint of suppressing the aggregation of compound (A) in the folding reaction solution, R １ ~R ３ The total number of carbon atoms is preferably 50 or less, more preferably 40 or less, even more preferably 30 or less, and even more preferably 26 or less. ４ ~R ６ The total number of carbon atoms is preferably 50 or less, more preferably 40 or less, even more preferably 30 or less, and even more preferably 26 or less.

[0030] R １ ~R ６ The aliphatic hydrocarbon group in R may be saturated or unsaturated, but saturated is preferred. １ ~R ６ The aliphatic hydrocarbon group in this compound may be linear or branched, but linear is preferred.

[0031] R １ ~R ６ The alkyl group is preferably a linear or branched alkyl group having 1 to 24 carbon atoms, and more preferably a linear alkyl group having 1 to 24 carbon atoms. The number of carbon atoms of the linear or branched alkyl group is preferably 1 to 20, more preferably 1 to 18, and even more preferably 1 to 16. １ ~R ６ Specific examples include methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, n-heptyl group, n-octyl group, n-nonyl group, n-decyl group, n-undecyl group, n-dodecyl group, n-tridecyl group, n-tetradecyl group, n-pentadecyl group, n-hexadecyl group, n-heptadecyl group, n-octadecyl group, n-nonadecyl group, n-icosyl group, n-henicosyl group, n-docosyl group, n-tricosyl group, n-tetracosyl group, and the like.

[0032] R １ ~R ６ These may be the same or different from each other. １ ~R ３ These may be the same or different from each other. ４ ~R ６ These may be the same or different from each other. １and R ４ They may be the same or different, but it is preferable that they be the same. ２ and R ５ They may be the same or different, but it is preferable that they be the same. ３ and R ６ They may be the same or different, but it is preferable that they be the same.

[0033] In the above formula (A), Y １ and Y ２ In this, the aliphatic hydrocarbon group having 1 to 10 carbon atoms, which may contain an oxygen atom, preferably has 1 to 8 carbon atoms, more preferably 1 to 6 carbon atoms, even more preferably 1 to 4 carbon atoms, and even more preferably 2 or 3 carbon atoms. １ and Y ２ The aliphatic hydrocarbon group in Y may be saturated or unsaturated, but saturated is preferred. １ and Y ２ The aliphatic hydrocarbon group in this expression may be linear or branched, but linear is preferred. "Aliphatic hydrocarbon group that may contain an oxygen atom" refers to an unsubstituted aliphatic hydrocarbon group or an aliphatic hydrocarbon group containing an oxygen atom. "Unsubstituted aliphatic hydrocarbon group" refers to an aliphatic hydrocarbon group composed only of carbon and hydrogen atoms. "Aliphatic hydrocarbon group containing an oxygen atom" refers to a group in which one or more methylene groups constituting the aliphatic hydrocarbon chain are substituted with oxygen atoms. １ and Y ２ For example, the number of oxygen atoms that may be included is 1 to 5.

[0034] Y １ and Y ２ The linear or branched alkylene group having 1 to 10 carbon atoms, or the linear or branched polyoxyalkylene group having 1 to 10 carbon atoms, is preferred. Specific examples of the polyoxyalkylene group include the polyoxyethylene group and the polyoxypropylene group. The number of carbon atoms in the linear or branched alkylene group and the linear or branched polyoxyalkylene group is preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 4, and even more preferably 2 or 3. １ and Y２ Preferably, it is a linear alkylene group. Y １ and Y ２ Specific examples of Y and Y include a methylene group, an ethylene group, an n-propylene group, an n-butylene group, an n-pentylene group, an n-hexylene group, an n-heptylene group, an n-octylene group, an n-nonylene group, an n-decylene group, and the like.

[0035] Y １ and Y ２ may be the same as or different from each other, but it is preferable that they are the same.

[0036] In the formula (A), n is an integer of 1 or more, and X ｎ－ represents an n-valent anion. X ｎ－ may be an organic anion or an inorganic anion, but an inorganic anion is preferable. X ｎ－ Preferably, it is a halide ion. X ｎ－ Specific examples of X include fluoride ion (F － ), chloride ion (Cl － ), bromide ion (Br － ), and iodide ion (I － ).

[0037] As the compound (A), a compound represented by the following general formula (A1) is preferable.

[0038] [In the formula, R １ to R ６ each independently represents an aliphatic hydrocarbon group having 1 to 24 carbon atoms. n is an integer of 1 or more, and X ｎ－ represents an n-valent anion. ]

[0039] R in the formula (A1) １ to R ６ , n, and X ｎ－ are the same as R １ to R ６ , n, and X ｎ－ in the formula (A).

[0040] As the compound (A), a compound represented by the following general formula (A1-1) is preferable.

[0041] [In the formula, R１ and R ４ Each of these independently represents an aliphatic hydrocarbon group having 1 to 24 carbon atoms. n is an integer greater than or equal to 1, and X ｎ－ This represents an n-valent anion.

[0042] R in the above formula (A1-1) １ , R ４ , n and X ｎ－ R in formula (A) １ , R ４ , n and X ｎ－ It is the same as this.

[0043] R １ and R ４ The number of carbon atoms in R may be selected according to the hydrophobicity of the target protein. For example, if the target protein has high hydrophobicity, １ and R ４ The number of carbon atoms in may be increased. For example, if the hydrophobicity of the target protein is low, R １ and R ４ The number of carbon atoms in this region may be reduced.

[0044] As compound (A), a compound represented by the following general formula (A1-1-1) is preferred.

[0045] [In the formula, m1 and m2 each independently represent integers from 1 to 24. n is an integer greater than or equal to 1, and X ｎ－ This represents an n-valent anion.

[0046] n and X in the above formula (A1-1-1) ｎ－ n and X in formula (A) are the same. ｎ－ It is the same as this.

[0047] From the viewpoint of suppressing the aggregation of compound (A) in the folding reaction solution, m1 and m2 are preferably 22 or less, more preferably 20 or less, even more preferably 18 or less, and even more preferably 16 or less. From the viewpoint of promoting the folding of the target protein, m1 and m2 are preferably 6 or more, 8 or more, 10 or more, 12 or more, or 14 or more. Examples of the ranges for m1 and m2 include 1-22, 1-20, 1-18, 1-16, 1-14, 6-24, 6-22, 6-20, 6-18, 6-16, 6-14, 8-24, 8-22, 8-20, 8-18, 8-16, 8-14, 10-24, 10-22, 10-20, 10-18, 10-16, 12-24, 12-22, 12-20, 12-18, 12-16, 14-24, 14-22, 14-20, 14-18, 14-16, etc. m1 and m2 may be selected according to the hydrophobicity of the target protein.

[0048] Specific examples of compound (A) include the compound represented by the following chemical formula (C16N-SS) (C16N-SS), the compound represented by the following chemical formula (C8N-SS) (C8N-SS), and the compound represented by the following chemical formula (MeN-SS) (MeN-SS).

[0049]

[0050] Compound (A) can be prepared by combining known methods, as described in the examples below. For example, it can be synthesized according to the methods described by G. J. L. Bernardes et al. (GJL Bernardes et al., J. Am. Chem. Soc. 2008, 130, 5052), R. Caraballo et al. (R. Caraballo et al., Chem. Commun. 2010, 46, 8469), and V. M. J. Saily et al. (VMJ Saily et al., Langmuir 2006, 22, 956), etc.

[0051] <Optional Components> The protein folding agent of this embodiment may contain other components in addition to compound (A). Examples of other components include oxidizing agents, reducing agents, surfactants, organic solvents, etc. Examples of reducing agents include compounds having a thiol group. Examples of reducing agents include reduced glutathione (GSH), 1,4-dithiothreitol, (±)-trans-1,2-bis(2-mercaptoacetamide)cyclohexane (BMC) (KJ Woycechowsky et al., Chem. Biol., 1999, 6, 871-879), aromatic thiols (DJ Madar et al., J. Biotech., 2009, 142, 214-219), cyclic selenoxide (K. Arai, K et al., Chem. Eur. J., 2011, 17, 481-485), and peptides having a Cys-XX-Cys structure (X is any amino acid, Cys is cysteine) (WJ Lees et al., Curr. Opin. Chem. Biol., 2008, 12, Examples include 740-745), selenoglutathione (J. Beld et al., Biochemistry. 2007 May 8;46(18):5382-90), selenol-containing peptides (S. Tsukagoshi et al., Chem. Asian J. 2020 September 1;15(17):2646-52), and thiol compounds described in Japanese Patent Publication No. 2022-135954. Examples of oxidizing agents include compounds having disulfide bonds other than compound (A). Examples of oxidizing agents include oxidized forms of the above reducing agents. Examples of surfactants are those described later. Organic solvents can be used to dissolve compound (A). For example, if the solubility of compound (A) in formula (A) or (A1) is low, an organic solvent may be used to dissolve compound (A). For example, R in formula (A) or (A1) １ ~R ６ If one or more of the elements have 3 to 24, 5 to 24, 6 to 24, or 8 to 24 carbon atoms, an organic solvent may be used. For example, R in formula (A1-1) １and R ４ If the carbon atom has 3 to 24, 5 to 24, 6 to 24, or 8 to 24 carbon atoms, an organic solvent may be used. Examples of organic solvents include dimethyl sulfoxide (DMSO).

[0052] The protein folding agent of this embodiment contains compound (A) as an active ingredient. Therefore, it can promote the folding of the target protein in a folding reaction solution containing the target protein at a high concentration of several hundred μM or more. Examples of the high concentration of the target protein include 200 μM or more, 300 μM or more, and 400 μM or more. The upper limit of the concentration of the target protein in the folding reaction solution should be any concentration at which the folding of the target protein proceeds. Examples of such concentrations of the target protein include 3000 μM or less, 2000 μM or less, 1000 μM or less, 800 μM or less, 700 μM or less, 600 μM or less, and 500 μM or less. The lower and upper limits can be combined arbitrarily. When using a higher concentration of the target protein than conventional methods, the concentration of the target protein in the folding reaction solution can be, for example, 200-3000 μM, 200-2000 μM, 200-1000 μM, 200-800 μM, 200-700 μM, 200-600 μM, 200-500 μM, 300-3000 μM, Examples of high concentrations include 300-2000 μM, 300-1000 μM, 300-800 μM, 300-700 μM, 300-600 μM, and 300-500 μM, 400-3000 μM, 400-2000 μM, 400-1000 μM, 400-800 μM, 400-700 μM, 400-600 μM, and 400-500 μM. The protein folding agent of this embodiment can also be used when the concentration of the target protein in the folding reaction solution is lower than the high concentrations mentioned above (for example, 1-200 μM). The concentration of the target protein can be appropriately selected depending on the type of target protein.

[0053] The protein folding agent of this embodiment can be used for folding unfolded proteins or refolding misfolded proteins. An unfolded protein is a protein that has not been folded. In unfolded proteins, the disulfide bonds formed in the natural structure of the protein are reduced to thiol groups. A protein that has been reduced and denatured by a reducing agent is an unfolded protein. A misfolded protein is a protein in which one or more disulfide bonds are formed in a structure other than the most stable natural structure. Unfolded proteins and misfolded proteins cannot express the activity possessed by the natural structure of the protein. By applying the protein folding agent of this embodiment to such unfolded or misfolded proteins, they can be folded into the most stable natural structure. The natural structure of a protein can also be said to be the three-dimensional structure in which the thermodynamic free energy is minimized.

[0054] The protein folding agent of this embodiment can promote the folding of target proteins in a folding reaction solution containing a high concentration of the target protein of several hundred μM or more. Therefore, it is possible to efficiently produce naturally structured proteins. The protein folding agent of this embodiment can also be used as a stabilizer when storing naturally structured proteins.

[0055] [Composition for Protein Folding] A second aspect of the present disclosure is a composition for protein folding. The composition for protein folding according to this aspect comprises a protein folding agent according to the first aspect and a protein aggregation inhibitor.

[0056] <Protein Folding Agent> As the protein folding agent, the protein folding agent according to the first embodiment described above can be used. One type of protein folding agent may be used alone, or two or more types may be used in combination.

[0057] <Protein Aggregation Inhibitors> A "protein aggregation inhibitor" refers to a drug that has the effect of suppressing the aggregation of the protein to be folded (target protein) in the folding reaction solution. By suppressing the aggregation of the target protein using a protein aggregation inhibitor, the folding of the target protein by compound (A) can be further promoted. It is preferable that the protein aggregation inhibitor does not denature the protein. When compound (A) has high hydrophobicity, the protein aggregation inhibitor also has the effect of suppressing the aggregation of compound (A) in aqueous solution. R in formula (A) or (A1) above １ ~R ６ The more carbon atoms there are, the more hydrophobic compound (A) becomes. For example, R in formula (A) or (A1) １ ~R ６ If one or more of the elements have 3 to 24, 5 to 24, 6 to 24, or 8 to 24 carbon atoms, the protein aggregation inhibitor may have the effect of suppressing the aggregation of compound (A).

[0058] Examples of protein aggregation inhibitors include surfactants. Surfactants inhibit the aggregation of target proteins by forming micelles containing the target protein in the internal phase in an aqueous solvent. Examples of surfactants include anionic surfactants, cationic surfactants, nonionic surfactants, and amphoteric surfactants.

[0059] Examples of anionic surfactants include carboxylic acid surfactants, sulfonic acid surfactants, sulfate ester surfactants, and phosphate ester surfactants. Examples of carboxylic acid surfactants include sodium octanoate, sodium decanoate, sodium laurate, sodium myristate, and sodium palmitate. Examples of sulfonic acid surfactants include sodium 1-hexanesulfonate, sodium 1-octanesulfonate, sodium 1-decanesulfonate, sodium 1-dodecanesulfonate, perfluorobutanesulfonic acid, trisodium naphthalenetrisulfonate, and sodium butylnaphthalenesulfonate. Examples of sulfate ester surfactants include sodium lauryl sulfate, sodium myristyl sulfate, sodium laureth sulfate, sodium polyoxyethylene alkylphenolsulfonate, and ammonium lauryl sulfate. Examples of phosphate ester surfactants include lauryl phosphate, sodium lauryl phosphate, and potassium lauryl phosphate.

[0060] Examples of cationic surfactants include quaternary ammonium salt surfactants, alkylamine salt surfactants, and pyridine surfactants. Examples of quaternary ammonium salt surfactants include tetramethylammonium chloride, tetramethylammonium hydroxide, tetrabutylammonium chloride, dodecyldimethylbenzylammonium chloride, alkyltrimethylammonium chloride, octyltrimethylammonium chloride, decyltrimethylammonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, cetyltrimethylammonium chloride, stearyltrimethylammonium chloride, alkyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, benzyltrimethylammonium chloride, benzyltriethylammonium chloride, benzalkonium chloride, benzalkonium bromide, benzethonium chloride, dialkyldimethylammonium chloride, didecyldimethylammonium chloride, and distearyldimethylammonium chloride. Examples of alkylamine salt surfactants include monomethylamine hydrochloride, dimethylamine hydrochloride, and trimethylamine hydrochloride. Examples of pyridine-based surfactants include butylpyridinium chloride, dodecylpyridinium chloride, and cetylpyridinium chloride.

[0061] Examples of nonionic surfactants include ester surfactants, ether surfactants, ester ether surfactants, alkanolamide surfactants, and alkyl glycoside surfactants. Examples of ester surfactants include glyceryl laurate, glyceryl monostearate, sorbitan fatty acid esters, and sucrose fatty acid esters. Examples of ether surfactants include polyoxyethylene alkyl ethers, pentaethylene glycol monododecyl ethers, octaethylene glycol monododecyl ethers, polyoxyethylene alkylphenyl ethers, octylphenol ethoxylate, nonylphenol ethoxylate, and polyoxyethylene polyoxypropylene glycol. Examples of ester ether surfactants include polyoxyethylene glycerin fatty acid esters, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene sorbitol fatty acid esters, polyoxyethylene hexitane fatty acid esters, and sorbitan fatty acid ester polyethylene glycol. Examples of alkanolamide surfactants include lauric acid diethanolamide, oleic acid diethanolamide, and stearate diethanolamide. Examples of alkyl glycoside surfactants include octyl glucoside, decyl glucoside, and lauryl glucoside.

[0062] Examples of amphoteric surfactants include alkyl betaine surfactants, fatty acid amidopropyl betaine surfactants, alkylimidazole surfactants, amino acid surfactants, and amine oxide surfactants. Examples of alkyl betaine surfactants include lauryldimethylaminoacetic acid betaine, stearyldimethylaminoacetic acid betaine, dodecylaminomethyldimethylsulfopropyl betaine, and octadecylaminomethyldimethylsulfopropyl betaine. Examples of fatty acid amidopropyl betaine surfactants include cocamidopropyl betaine and cocamidopropyl hydroxysultaine. Examples of alkylimidazole surfactants include 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine. Examples of amino acid surfactants include sodium lauroyl glutamate, potassium lauroyl glutamate, and lauroylmethyl-β-alanine. Examples of amine oxide surfactants include lauryldimethylamine N-oxide and oleyldimethylamine N-oxide.

[0063] Cationic surfactants are preferred as protein aggregation inhibitors, and quaternary ammonium salt surfactants are more preferred. Examples of quaternary ammonium salt surfactants include compounds represented by the following general formula (B) (hereinafter also referred to as "compound (B)").

[0064] [In the formula, Rb １ ~Rb ４ Each of these independently represents a hydrocarbon group with 1 to 24 carbon atoms. n is an integer greater than or equal to 1, and X ｎ－ This represents an n-valent anion.

[0065] n and X in formula (B) above ｎ－ n and X in formula (A) are the same. ｎ－ It is the same as this.

[0066] In the above formula (B), Rb １ ~Rb ４ The hydrocarbon group having 1 to 24 carbon atoms in this compound may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group.

[0067] The aforementioned aromatic hydrocarbon group is a hydrocarbon group having at least one aromatic ring. Specific examples of aromatic rings include benzene rings, naphthalene rings, anthracene rings, phenanthrene rings, and the like. Rb １ ~Rb ４ Examples of aromatic hydrocarbon groups include groups obtained by removing one hydrogen atom from an aromatic ring (aryl group), and groups in which one hydrogen atom of an aromatic ring is replaced by an alkylene group (alkylaryl group). Examples of alkyl groups in the alkylaryl group include linear or branched alkyl groups having 1 to 10 carbon atoms. Examples of the aryl group include phenyl group and naphthyl group, with phenyl group being preferred. Examples of the alkylaryl group include benzyl group and phenethyl group, with benzyl group being preferred.

[0068] Rb １ ~Rb ４ The aliphatic hydrocarbon group in Rb may be saturated or unsaturated, but saturated is preferred. １ ~Rb ４６ The aliphatic hydrocarbon group in Rb may be linear or branched, but linear is preferred. １ ~Rb ４ The number of carbon atoms in the aliphatic hydrocarbon group in Rb is preferably 22 or less, more preferably 20 or less, even more preferably 18 or less, and even more preferably 16 or less, from the viewpoint of suppressing aggregation of compound (B) in the folding reaction solution. １ ~Rb ４ Examples of the range of carbon atoms in the aliphatic hydrocarbon group in Rb include 1-22, 1-20, 1-18, 1-16, etc. １ ~Rb ４ The number of carbon atoms in the aliphatic hydrocarbon group may be selected according to the hydrophobicity of the target protein.

[0069] From the perspective of suppressing the aggregation of target proteins, Rb １ and Rb ４ In Rb, the aliphatic hydrocarbon group preferably has 6 or more carbon atoms, 8 or more, 10 or more, 12 or more, or 14 or more. １ and Rb ４Examples of the number of carbon atoms in the aliphatic hydrocarbon group in Rb include 1-24, 6-24, 8-24, 10-24, 12-24, 14-24, 16-24, 1-20, 6-20, 8-20, 10-20, 12-20, 14-20, 16-20, 1-18, 6-18, 8-18, 10-18, 12-18, 14-18, 16-18, 1-16, 6-16, 8-16, 10-16, 12-16, 14-16, etc. ２ , Rb ３ , Rb ５ , and Rb ６ In Rb, the aliphatic hydrocarbon group preferably has 10 or fewer carbon atoms, 8 or fewer, 6 or fewer, 4 or fewer, or 2 or fewer. ２ , Rb ３ , Rb ５ , and Rb ６ Examples of the number of carbon atoms in the aliphatic hydrocarbon group in Rb include 1 to 10, 1 to 8, 1 to 6, 1 to 4, and 1 to 2. In some embodiments, Rb ２ , Rb ３ , Rb ５ , and Rb ６ The number of carbon atoms is 1 or 2.

[0070] From the viewpoint of suppressing the aggregation of compound (B) in the folding reaction solution, Rb １ ~Rb ４ The total number of carbon atoms is preferably 50 or less, more preferably 40 or less, even more preferably 30 or less, and even more preferably 26 or less.

[0071] Rb １ ~Rb ４ The aliphatic hydrocarbon group in is preferably a linear or branched alkyl group having 1 to 24 carbon atoms, and more preferably a linear alkyl group having 1 to 24 carbon atoms. Rb １ ~Rb ４ A specific example of an aliphatic hydrocarbon group in the above general formula (A) is R １ ~R ６ Similar examples include the above.

[0072] Rb １ ~Rb ４The group is preferably a linear or branched aliphatic hydrocarbon group having 1 to 24 carbon atoms, more preferably a linear or branched alkyl group having 1 to 24 carbon atoms, and even more preferably a linear alkyl group having 1 to 24 carbon atoms.

[0073] Rb １ ~Rb ３ They may be the same or different from one another.

[0074] As compound (B), the compound represented by the following general formula (B1-1) is preferred.

[0075] [In the formula, Rb １ represents a hydrocarbon group with 1 to 24 carbon atoms. n is an integer greater than or equal to 1, and X ｎ－ This represents an n-valent anion.

[0076] Rb in the above formula (B1-1) １ , n and X ｎ－ Rb in formula (B) １ , n and X ｎ－ It is the same as this.

[0077] Specific examples of compound (B) include the compounds listed above as quaternary ammonium salt-based surfactants. Among these, alkyltrimethylammonium bromide is preferred as compound (B). Specific examples of alkyltrimethylammonium bromide include hexadecyltrimethylammonium bromide (CTAB), n-octyltrimethylammonium bromide, dodecyltrimethylammonium bromide, trimethylstearylammonium bromide, and the like. In some embodiments, compound (B) is CTAB.

[0078] Protein aggregation inhibitors may be used individually or in combination of two or more.

[0079] Examples of molar ratios of compound (A) to protein aggregation inhibitor in the protein folding composition include 1:1 to 1:500, 1:1 to 1:300, 1:5 to 1:300, 1:10 to 1:300, 1:15 to 1:300, 1:20 to 1:300, 1:25 to 1:300, 1:30 to 1:300, 1:1 to 1:200, 1:5 to 1:200, 1:10 to 1:200, 1:15 to 1:200, 1:20 to 1:200, 1:25 to 1:200, and 1:30 to 1:200.

[0080] <Optional Components> The protein folding composition of this embodiment may contain other components in addition to the components listed above. Examples of other components include, but are not limited to, solvents, diluents, vehicles, excipients, flow promoters, binders, granulators, and stabilizers. The other components may be used individually or in combination of two or more.

[0081] The protein folding composition of this embodiment contains a protein aggregation inhibitor in addition to compound (A), thereby further promoting the folding of the target protein in a folding reaction solution containing a high concentration of the target protein. The target protein concentration to which the protein folding composition of this embodiment is applied is the same as the concentration listed in the [Protein Folding Agent] section above.

[0082] [Protein Folding Kit] A third aspect of the present disclosure is a protein folding kit. The protein folding kit according to this aspect includes the protein folding agent according to the first aspect.

[0083] As the protein folding agent, the protein folding agent according to the first embodiment described above can be used. The protein folding agent may be used alone or in combination of two or more types.

[0084] <Optional Configurations> In addition to the above configurations, the protein folding kit may include other components. Examples of other components include protein aggregation inhibitors, polar organic solvents, buffer solutions, folding reaction containers, and instructions.

[0085] (Protein Aggregation Inhibitors) The protein aggregation inhibitors listed in the section above [Compositions for Protein Folding] may be used. One type of protein aggregation inhibitor may be used alone, or two or more types may be used in combination.

[0086] (Polar Organic Solvents) Polar organic solvents can be added to the folding reaction solution after the folding reaction. By adding a polar solvent after the folding reaction, the naturally structured protein produced by folding can be stably maintained. This can increase the amount of naturally structured protein obtained after the folding reaction. Polar organic solvents are thought to have the effects of compound (A), a protein aggregation inhibitor, and the dissociation of micelles, which are aggregates of the target protein. Naturally structured proteins formed by folding generally have lower hydrophobicity compared to the denatured target protein before folding. Therefore, it is thought that the naturally structured protein is released into the aqueous phase by the collapse of micelles and is stably maintained in the aqueous phase.

[0087] Examples of polar organic solvents include nitrile organic solvents, alcohol organic solvents, ketone organic solvents, ether organic solvents, ester organic solvents, amide organic solvents, and sulfoxide organic solvents. Examples of nitrile organic solvents include acetonitrile, benzonitrile, butyronitrile, 3-methoxypropionitrile, propionitrile, 3,3'-oxydipropionitrile, methoxyacetonitrile, pimelonitrile, phenylacetonitrile, and glutaronitrile. Examples of alcohol organic solvents include aliphatic alcohols. Examples of aliphatic alcohols include methanol, ethanol, 1-propanol, isopropanol, 1-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl-2-propanol, ethylene glycol, and glycerin. Examples of ketone organic solvents include acetone, 2-butanone, and 4-methyl-2-pentanone. Examples of ether organic solvents include diethyl ether, methoxycyclopentane, tetrahydrofuran, triethylene glycol dimethyl ether, and dibutyl ether. Examples of ester-based organic solvents include ethyl acetate, butyl acetate, and isobutyl acetate. Examples of amide-based organic solvents include dimethylformamide, diethylformamide, and dimethylacetamide. Examples of sulfoxide-based organic solvents include dimethyl sulfoxide and di-n-butyl sulfoxide.

[0088] (Buffer) A buffer can be used to prepare the folding reaction solution. The buffer is not particularly limited as long as it allows the folding reaction of the target protein to proceed, and any buffer commonly used in the field of biochemistry can be used without particular restriction. Examples of buffers include amine-based buffers such as Tris-HCl buffer, Tris buffer, MES buffer, and Trisine buffer; phosphate buffer; and Good's buffers. The pH of the buffer is typically pH 4 to 10. The pH of the buffer is preferably pH 5 to 9, more preferably pH 7 to 9, and even more preferably pH 7 to 8. A Tris-HCl buffer with added sodium chloride is preferred as a buffer. A specific example of a buffer is a buffer containing 50 mM tris-hydroxymethylaminomethane and 300 mM sodium chloride, adjusted to pH 7.5 with hydrochloric acid.

[0089] The kit of this embodiment may include a concentrated buffer solution. The kit of this embodiment may include reagents for preparing the buffer solution.

[0090] The kit of this embodiment can be used to fold the target protein.

[0091] [Method for Folding Proteins] A fourth aspect of this disclosure is a method for folding proteins. The method for folding proteins according to this aspect includes step (a) of incubating a protein in the presence of a protein folding agent according to the first aspect.

[0092] <Step (a)> In step (a), the target protein is incubated in the presence of the protein folding agent according to the first embodiment.

[0093] The incubation of the target protein can be carried out by preparing a folding reaction solution by dissolving the target protein and the protein folding agent according to the first embodiment in a suitable buffer solution and then incubating it. By performing this incubation, the protein folding reaction can be carried out.

[0094] The buffer solution is not particularly limited as long as it allows the protein folding reaction to proceed; any buffer solution commonly used in biochemistry can be used without any restrictions. The same buffer solutions listed in the [Protein Folding Kit] section above can be used.

[0095] The concentration of the protein folding agent in the folding reaction solution is not particularly limited. As an example of the concentration of compound (A) in the folding reaction solution, it can be 1 μM to 500 mM. The concentration of compound (A) in the folding reaction solution is preferably 10 μM to 100 mM, more preferably 300 μM to 50 mM, even more preferably 500 μM to 10 mM, even more preferably 800 μM to 5 mM, and particularly preferably 1 mM to 3 mM.

[0096] Target proteins include those whose native structural form has one or more disulfide bonds. The number of disulfide bonds in the native structural form of the target protein is not particularly limited, as long as there is one or more. Examples of the number of disulfide bonds in the native structural form of the target protein include 1 to 20, 1 to 15, 1 to 10, and 1 to 5.

[0097] The molecular weight of the target protein is not particularly limited, but examples include 1,000 to 10,000,000, and preferably 1,000 to 250,000.

[0098] Unfolding proteins or misfolding proteins may be used as the target proteins. For example, the target protein may be a protein that has been reduced and denatured with a reducing agent.

[0099] Examples of target protein concentrations in the folding reaction solution include 1 to 3000 μM. Compound (A) can promote the folding of the target protein in a folding reaction solution containing a higher concentration of the target protein than conventional methods. Therefore, the target protein concentration in the folding reaction solution may be, for example, 200 μM or more, 300 μM or more, or 400 μM or more. The concentration of the target protein in the folding reaction solution may be, for example, 3000 μM or less, 2000 μM or less, 1000 μM or less, 800 μM or less, 700 μM or less, 600 μM or less, or 500 μM or less. The lower and upper limits can be combined arbitrarily. The concentration of the target protein in the folding reaction solution may be, for example, 200-3000 μM, 200-2000 μM, 200-1000 μM, 200-800 μM, 200-700 μM, 200-600 μM, 200-500 μM, 300-3000 μM, 300-2000 μM, 300-1000 μM, 300-800 μM, 300-700 μM, 300-600 μM, or 300-500 μM, 400-3000 μM, 400-2000 μM, 400-1000 μM, 400-800 μM, 400-700 μM, 400-600 μM, and 400-500 μM. Proteins with high water solubility can be used at higher concentrations.

[0100] The folding reaction solution may further contain a protein aggregation inhibitor. The protein aggregation inhibitor may be one of those listed in the section on [Compositions for Protein Folding] above. Step (a) may be a step of incubating the target protein in the presence of the protein folding agent and the protein aggregation inhibitor according to the first embodiment. In one embodiment, the protein aggregation inhibitor is a surfactant. In one embodiment, the protein aggregation inhibitor is a cationic surfactant. In one embodiment, the protein aggregation inhibitor is a quaternary ammonium salt surfactant. In one embodiment, the protein aggregation inhibitor is compound (B). In compound (B), Rb in general formula (B) １ ~Rb ４The protein aggregation inhibitor can be selected according to the hydrophobicity of the target protein. In one embodiment, the protein aggregation inhibitor is CTAB. The protein aggregation inhibitor may be used alone or in combination of two or more types.

[0101] When a protein flocculant inhibitor is used, the concentration of the protein flocculant in the folding reaction solution can be, for example, 1 to 500 mM. The concentration of the protein flocculant in the folding reaction solution may also be 5 to 300 mM, 10 to 300 mM, 20 to 300 mM, 25 to 300 mM, 30 to 300 mM, 5 to 200 mM, 10 to 200 mM, 20 to 200 mM, 25 to 200 mM, 30 to 200 mM, 5 to 100 mM, 10 to 100 mM, 20 to 100 mM, 25 to 100 mM, or 30 to 100 mM. In one embodiment, the concentration of the protein flocculant in the folding reaction solution may be 50 to 100 mM.

[0102] The folding reaction solution may further contain an organic solvent. The organic solvent can be used to dissolve compound (A). For example, if compound (A) has low solubility in water, an organic solvent may be used to dissolve compound (A). For example, R in formula (A) or (A1) １ ~R ６ If one or more of the elements have 3 to 24, 5 to 24, 6 to 24, or 8 to 24 carbon atoms, an organic solvent may be used. For example, R in formula (A1-1) １ and R ４ If the carbon atom has 3 to 24, 5 to 24, 6 to 24, or 8 to 24 carbon atoms, an organic solvent may be used. Examples of organic solvents include dimethyl sulfoxide (DMSO).

[0103] When using an organic solvent, the concentration of the protein flocculant in the folding reaction solution can be, for example, 0.1 to 10% by mass, or it may be 0.5 to 8% by mass, or 1 to 6% by mass.

[0104] The incubation temperature is not particularly limited as long as it is a temperature at which the folding reaction of the target protein can proceed. The incubation temperature can be appropriately selected depending on the type of target protein. For example, the incubation temperature can be 10 to 50°C, preferably 20 to 40°C, and more preferably 25 to 35°C. The incubation time can be appropriately selected depending on the type of target protein. For example, the incubation time can be 1 minute to 300 hours. The incubation time may be, for example, 1 minute or more, 10 minutes or more, 30 minutes or more, 60 minutes or more, 120 minutes or more, or 180 minutes or more. The incubation time may be, for example, 250 hours or less, 200 hours or less, 150 hours or less, 120 hours or less, 100 hours or less, 80 hours or less, 400 minutes or less, 300 minutes or less, 240 minutes or less, or 200 minutes or less. The upper and lower limits can be combined arbitrarily. Examples of incubation times include 10 minutes to 300 hours, 30 minutes to 300 hours or less, 60 minutes to 300 hours or less, 120 minutes to 300 hours or less, 10 minutes to 200 hours, 30 minutes to 200 hours or less, 60 minutes to 200 hours or less, 120 minutes to 200 hours or less, 10 minutes to 100 hours, 30 minutes to 100 hours or less, 60 minutes to 100 hours or less, 120 minutes to 100 hours or less, 10 to 400 minutes, 30 to 300 minutes, 60 to 240 minutes, and 120 to 200 minutes. The folding state of the target protein can be observed over time, and incubation may be terminated when the most stable native structure is achieved. For observation of the folding state, for example, separation by high-performance liquid chromatography (HPLC) analysis can be used. For example, reversed-phase HPLC can be used for HPLC analysis. For example, incubation may be terminated when the proportion of the most stable natural structural protein reaches a predetermined proportion or higher. Examples of the predetermined proportion include 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, and 95% or more.

[0105] <Optional Steps> The method of this embodiment may include optional steps in addition to the incubation step. Examples of optional steps include adding a polar organic solvent, unfolding the protein, and isolating the folded protein.

[0106] (Addition of polar organic solvent: step (b)) The method of this embodiment may include a step of adding a polar organic solvent to the folding reaction solution after step (a) (hereinafter also referred to as "step (b)"). By performing step (b) after step (a), the natural structural protein can be stably maintained and the efficiency of obtaining the natural structural protein can be increased.

[0107] Examples of polar organic solvents include those listed in the [Protein Folding Kit] section above. In one embodiment, the polar organic solvent is a nitrile-based organic solvent. In another embodiment, the polar organic solvent is acetonitrile. One type of polar organic solvent may be used alone, or two or more types may be used in combination.

[0108] The amount of polar organic solvent added is 6 to 19 μL per 100 μL of the folding reaction solution after step (a) (the mixture obtained in step (a)), preferably 7 to 18 μL, more preferably 7 to 17 μL, even more preferably 8 to 16 μL, and particularly preferably 10 to 15 μL.

[0109] Hydrochloric acid may be added to the folding reaction solution after step (a). Hydrochloric acid can be added to the folding reaction solution after step (a) so that the final concentration is 0.1 to 10 M, preferably 0.5 to 5 M, and more preferably 0.7 to 2 M. When hydrochloric acid is added to the folding reaction solution after step (a), the amount of polar organic solvent to be added is 6 to 19 μL per 100 μL of the mixture after hydrochloric acid is added, preferably 7 to 18 μL, more preferably 7 to 17 μL, even more preferably 8 to 16 μL, and particularly preferably 10 to 15 μL. Hydrochloric acid can be added to stop the folding reaction. If the folding reaction is not to be stopped, hydrochloric acid does not need to be added. Acids other than hydrochloric acid may be used to stop the folding reaction. For example, trifluoroacetic acid may be used.

[0110] Step (b) can be carried out at room temperature (10 to 40°C, preferably 15 to 30°C). The mixture after step (b) may be stored as a sample containing natural structural proteins. Examples of storage temperatures include 10 to 40°C.

[0111] (Protein Unfolding Step) The method of this embodiment may include a step of unfolding the protein before the incubation step (protein folding step). The protein unfolded by the unfolding step may be used as the target protein. The step of unfolding the protein can be carried out by incubating the protein in the presence of a reducing agent. Examples of reducing agents include reduced glutathione (GSH), β-mercaptoethanol, and 1,4-dithiothreitol. Protein denaturants (e.g., guanidine hydrochloride, urea, etc.) may be used for protein unfolding. The protein unfolding reaction may be carried out by preparing a protein unfolding reaction solution by dissolving the protein, reducing agent, and protein denaturant in a medium such as water or a buffer, and then incubating it. Examples of the protein concentration in the unfolding reaction solution include 0.1 to 100 mg / mL. Examples of the reducing agent concentration in the unfolding reaction solution include 0.1 to 1000 mM. The concentration of the oxidizing agent is preferably 1 to 500 mM, more preferably 10 to 300 mM, and even more preferably 50 to 200 mM. The concentration of the denaturing agent in the unfolding reaction solution is, for example, 0.1 to 100 M. The concentration of the denaturing agent is preferably 0.5 to 50 M, more preferably 1 to 30 M, and even more preferably 1 to 20 M. The pH of the unfolding reaction solution is, for example, pH 5 to 10, preferably pH 6 to 9.5, more preferably pH 7 to 9, and even more preferably pH 8 to 9.

[0112] Examples of buffers that can be used in the unfolding reaction solution are the same as those mentioned above. A specific example of a buffer is 0.1 M Tris-HCl buffer.

[0113] The incubation temperature is not particularly limited as long as it is a temperature at which the protein unfolding reaction can proceed. The incubation temperature can be appropriately selected depending on the type of protein. For example, the incubation temperature can be 10 to 60°C, preferably 20 to 55°C, and more preferably 20 to 50°C. The incubation time can be appropriately selected depending on the type of protein. For example, the incubation time can be 1 to 600 minutes, preferably 10 to 500 minutes, more preferably 30 to 400 minutes, and even more preferably 60 to 300 minutes. The unfolding state can be observed over time, and the incubation may be terminated when all disulfide bonds have been reduced.

[0114] After incubation, the unfolding reaction solution may be dialysis or otherwise performed to remove denaturing agents and reducing agents.

[0115] (Protein Isolation Step) The method of this embodiment may include a step of isolating the folded protein (natural structural protein) after the incubation step (protein folding step). Known protein isolation methods can be used for protein isolation without particular limitations. Examples of protein isolation methods include salting out, dialysis, column chromatography, etc.

[0116] In the method of this embodiment, compound (A) is used as a protein folding agent, allowing for efficient folding of the target protein using a folding reaction solution containing a high concentration of the target protein. By applying the method of this embodiment, an improvement in the efficiency of producing naturally structured proteins is expected. Therefore, it is useful in the production of protein products such as protein pharmaceuticals.

[0117] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0118] [Synthesis Example of Compound (1)] <Synthesis Example of Compound (MeN-SS)> Compound (MeN-SS) was synthesized according to previously reported methods (GJL Bernardes et al., J. Am. Chem. Soc. 2008, 130, 5052; R. Caraballo et al., Chem. Commun. 2010, 46, 8469). The synthesis scheme for compound (MeN-SS) is shown below.

[0119]

[0120] Compound 1 (3.08 g, 19.5 mmol) and sodium thiosulfate pentahydrate (4.74 g, 19.1 mmol) were mixed and dissolved in MilliQ (30.0 mL), and stirred overnight under a nitrogen atmosphere with reflux. The resulting reaction solution was dried under reduced pressure, 6 M hydrochloric acid (30.0 mL) was added, and the mixture was stirred at 85°C for 2 hours. The post-reaction solution was dried under reduced pressure, and dichloromethane (150 mL) was added to the resulting residue, and ethanol was added dropwise until the supernatant was no longer turbid. Sodium chloride and sulfate were removed from the resulting suspension by suction filtration, and the filtrate was dried under reduced pressure. The resulting residue was recrystallized with ethanol to obtain a solid. Saturated iodine-ethanol solution was added to the obtained solid until the reaction solution turned yellow, and the mixture was stirred at room temperature for 5 minutes. The post-reaction solution was dried under reduced pressure, and recrystallized with ethanol to obtain compound (MeN-SS). The obtained compound (MeN-SS) is, １ H NMR measurement (400MHz, D ２ It was identified by O, 295K (Figure 1) and ESI-TOF mass spectrometry (measurement mode: Positive, solvent: MilliQ; Figure 2).

[0121] <Synthesis Example of Compound (C16N-SS)> The synthesis scheme for compound (C16N-SS) is shown below. Compound 3 and compound (C16N-SS) were synthesized based on previously reported information (VMJ Saily et al., Langmuir 2006, 22, 956).

[0122]

[0123] (Synthesis of Compound 3) Compound 2 (1.61 g, 5.73 mmol) was dissolved in anhydrous methanol (16.0 mL), and then triethylamine (1.16 g, 11.5 mmol) was slowly added dropwise. After stirring at room temperature under a nitrogen atmosphere for 30 minutes, the reaction solution was dried under reduced pressure to obtain Compound 3.

[0124] (Synthesis of compound (C16N-SS)) Compound 3 was dissolved in 2-propanol (11.2 mL), and 1-iodohexadecane (8.08 g, 22.9 mmol) was slowly added dropwise. After stirring at room temperature under a nitrogen atmosphere for two weeks, the resulting suspension was filtered by suction. The filtrate was dried under reduced pressure, and the resulting residue was purified by silica gel chromatography (NH silica, developing solvent: chloroform / methanol = 9 / 1, 0.5% triethylamine) and recrystallization with ethanol to obtain compound (C16N-SS). The obtained compound (C16N-SS) is, １ H NMR measurement (400MHz, CDCl ３ , 297K; Figure 3), １３ C NMR measurement (400MHz, CDCl ３ It was identified by 298 K (Figure 4) and MALDI-TOF mass spectrometry (measurement mode: Reflector Positive, matrix: 2,5-dihydroxybenzoic acid; Figure 5).

[0125] (Reagents) (2-chloroethyl)trimethylammonium chloride (compound 1), triethylamine, 1-iodohexadecane, and 2,5-dihydroxybenzoic acid were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Sodium thiosulfate pentahydrate, hydrochloric acid, and 2-propanol were purchased from Kishida Chemical Co., Ltd. (Tokyo, Japan). Iodine, bis[2-(N,N-dimethylamino)ethyl]disulfide dihydrochloride (compound 2) were purchased from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). Dehydrated ethanol was purchased from Kanto Chemical Co., Ltd.

[0126] (Equipment) For NMR measurements, a JEOL Ltd. JNM-ECX400 instrument was used. MALDI-TOF mass spectrometry spectroscopy was performed using a Bruker K.K. autotoflex speed instrument. ESI-TOF mass spectrometry spectroscopy was performed using a Bruker K.K. microOTOF-QII instrument.

[0127] [Experimental Example 1] <Protein Aggregation Inhibition Test and Folding Test> Bovine Pancreatic Trypsin Inhibitor (BPTI), which has three disulfide bonds in its molecule, was used as a model protein.

[0128] (Preparation of reduced-modified BPTI) BPTI (10 mg) was dissolved in 0.1 M Tris-HCl (pH 8.0, 1.0 mL) containing 30 mM 1,4-dithiothreitol and 8 M urea, incubated at 50°C for 3 hours, and then purified by reverse-phase HPLC. HPLC purification was performed using a PU-4180 pump and UV-4075 detector manufactured by JASCO Corporation (Tokyo, Japan), and an InertSustain C18 column (4.6 mm × 250 mm) manufactured by GL Sciences Co., Ltd. (Tokyo, Japan), at a flow rate of 1.0 mL / min. －１ The procedure was carried out as follows. The recovered fraction was freeze-dried, and the resulting powder was stored at -30°C.

[0129] (Reagents) BPTI was purchased from Pro-Spec-Tany TechnoGene Ltd. (Rehovot, Israel). 1,4-Dithiothreitol and urea were purchased from Nacalai Tesque Co., Ltd. (Kyoto, Japan). Hexadecyltrimethylammonium bromide (CTAB), n-Octyltrimethylammonium bromide (C8TAB), and dodecyltrimethylammonium bromide (C12TAB) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).

[0130] (Equipment) For the analysis of the folding process using reverse-phase HPLC, a Primade HPLC system manufactured by Hitachi High-Tech Corporation (Tokyo, Japan) and a TSKgel Protein C4-300 column (φ4.6 × 150 mm) manufactured by Tosoh Corporation (Tokyo, Japan) were used.

[0131] (Evaluation of the protein aggregation inhibitory effect of ammonium lipids) In the presence of 50 mM ammonium lipid (C8TAB, C12TAB, or CTAB), reductively denatured BPTI (400 μM) was dissolved in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) and incubated at 30°C. Photographs were taken after incubation for 3 hours. For comparison, photographs were also taken of a sample in which reductively denatured BPTI (400 μM) was dissolved in Tris-HCl buffer and incubated at 30°C for 3 hours in the absence of ammonium lipids.

[0132] The results are shown in Figure 6. In Figure 6, "C16TAB" refers to CTAB. Aggregation of BPTI was observed in the sample without ammonium lipids, the sample with C8TAB added, and the sample with C12TAB added. In the sample with CTAB (C16TAB) added, neither BPTI nor CTAB aggregation was observed. These results confirm that CTAB is effective in suppressing BPTI aggregation. The type of ammonium lipid effective in suppressing protein aggregation is thought to be influenced by the hydrophobicity of the protein, etc. Therefore, it is thought that there are proteins for which C8TAB or C12TAB is effective in suppressing aggregation.

[0133] (Folding test using CTAB) In the presence of 50 mM CTAB, reduced-modified BPTI (400 μM) was dissolved in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) and incubated at 30°C. Reaction solutions (25.2 μL) were sampled at predetermined times (0 min, 10 min, 30 min, 60 min, 120 min, 180 min) and mixed with 1 M hydrochloric acid (374.8 μL), and analyzed by reverse-phase HPLC. Flow rate: 1.0 mL / min －１The reaction was tracked at a wavelength of 229 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: water containing 0.05% trifluoroacetic acid (TFA), solvent B: acetonitrile containing 0.05% TFA; concentration of solvent A: 95% at 0 min, 80% at 15 min, 65% at 45 min). Hydrochloric acid was added to stop the folding reaction.

[0134] The results are shown in Figure 7. The reduced-denatured BPTI remained largely unfolded even after incubation for 180 minutes.

[0135] (Folding test using CTAB and compound (MeN-SS)) Reducible BPTI (400 μM) was dissolved in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) in the presence of 50 mM CTAB and 1.2 mM compound (MeN-SS), and incubated at 30°C. Reaction solutions (25.2 μL) were sampled at predetermined times (0 min, 10 min, 30 min, 60 min, 120 min, 180 min) and mixed with 1 M hydrochloric acid (374.8 μL). Analysis was performed by reverse-phase HPLC. Flow rate: 1.0 mL / min －１ The process was tracked at a wavelength of 229 nm, and a linear gradient was applied to the solvent ratio during measurement (solvent A: water containing 0.05% TFA, solvent B: acetonitrile containing 0.05% TFA; concentration of solvent A: 95% at 0 minutes, 80% at 15 minutes, 65% at 45 minutes).

[0136] The results are shown in Figure 8. It was confirmed that, over time, the reduced BPTI folded, and the natural structure BPTI was formed.

[0137] (Folding test using CTAB and compound (C16N-SS)) Reducible BPTI (400 μM) was dissolved in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) in the presence of 50 mM CTAB and 1.2 mM compound (C16N-SS), and incubated at 30°C. Reaction solutions (25.2 μL) were sampled at predetermined times (0 min, 10 min, 30 min, 60 min, 120 min, 180 min) and mixed with 1 M hydrochloric acid (374.8 μL). The mixture was analyzed by reverse-phase HPLC. Flow rate: 1.0 mL / min－１ The process was tracked at a wavelength of 229 nm, and a linear gradient was applied to the solvent ratio during measurement (solvent A: water containing 0.05% TFA, solvent B: acetonitrile containing 0.05% TFA; concentration of solvent A: 95% at 0 minutes, 80% at 15 minutes, 65% at 45 minutes).

[0138] The results are shown in Figure 9. It was confirmed that, over time, the reduction-modified BPTI folded, and the natural structure BPTI was formed. Compared to the case using compound (MeN-SS), the rate of formation of the natural structure BPTI was faster and the proportion of the BPTI formed was higher when compound (C16N-SS) was used.

[0139] (Evaluation of the effect of CTAB concentration on folding tests) Reducible BPTI (400 μM) was dissolved in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) in the presence of 0–300 mM CTAB and 1.2 mM compound (C16N-SS), and incubated at 30°C. Three hours after the start of the reaction, a fraction of the reaction solution (25.2 μL) was mixed with 1 M hydrochloric acid (374.8 μL) and analyzed by reverse-phase HPLC. Flow rate: 1.0 mL / min －１ The process was tracked at a wavelength of 229 nm, and a linear gradient was applied to the solvent ratio during measurement (solvent A: water containing 0.05% TFA, solvent B: acetonitrile containing 0.05% TFA; concentration of solvent A: 95% at 0 minutes, 80% at 15 minutes, 65% at 45 minutes).

[0140] The results are shown in Figure 10. The proportion of BPTI forming its native structure in each plot in Figure 10 was calculated by taking the ratio of the peak area of ​​the native structure (N) to the peak area of ​​the reduced-modified state (R) immediately after the start of the reaction in each chromatogram obtained at each CTAB concentration. When the CTAB concentration was 50 mM and 100 mM, the proportion of BPTI forming its native structure was 70% or more. These results confirm that the folding efficiency of BPTI is high when the CTAB concentration is between 50 and 100 mM.

[0141] (Evaluation of the effect of acetonitrile addition after incubation) Reducible BPTI (400 μM) was dissolved in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) in the presence of 50 mM CTAB and 1.2 mM compound (C16N-SS), and incubated at 30°C. Three hours after the start of the reaction, a fraction of the reaction solution (25.2 μL) was mixed with 1 M hydrochloric acid (374.8 μL), and acetonitrile was added to the mixture at predetermined ratios (5 v / v%, 10 v / v%, 15 v / v%, 20 v / v%, 30 v / v%, 40 v / v%, or 50 v / v%). The resulting solution was analyzed by reverse-phase HPLC. Flow rate: 1.0 mL / min －１ The process was tracked at a wavelength of 229 nm, and a linear gradient was applied to the solvent ratio during measurement (solvent A: water containing 0.05% TFA, solvent B: acetonitrile containing 0.05% TFA; concentration of solvent A: 95% at 0 minutes, 80% at 15 minutes, 65% at 45 minutes).

[0142] The results are shown in Figure 11. The proportion of BPTI formed into its natural structure in each plot in Figure 11 was calculated by taking the ratio of the peak area of ​​the natural structure (N) to the peak area of ​​the reduced-modified state (R) immediately after the start of the reaction in each chromatogram obtained at each CTAB concentration. When 10 v / v% and 15 v / v% acetonitrile were added after incubation, the proportion of BPTI formed into its natural structure could be increased. Acetonitrile is thought to have the effect of dissociating micelles formed by BPTI, CTAB, and the compound (C16N-SS). It is presumed that 10 to 20 v / v%, preferably 10 to 15 v / v%, of acetonitrile contributes to the stable maintenance of the natural structure BPTI by dissociating micelles containing the natural structure BPTI.

[0143] (Evaluation of the protein aggregation inhibitory effect of compound (C16N-SS)) Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) containing CTAB, compound (C16N-SS), and dimethyl sulfoxide (DMSO) was prepared by adding urea and reduced-denatured BPTI dissolved in Tris-HCl buffer. The final concentrations of each component were CTAB 50 mM, compound (C16N-SS) 1.2 mM, DMSO 3% (w / w), urea 246 mM, and reduced-denatured BPTI 400 μM. This was incubated at 30°C. Photographs were taken after incubation for 3 hours. In addition, the incubation was performed at 30°C using the same method as above, except that oxidized glutathione (GSSG) was used instead of compound (C16N-SS) and CTAB was not used. After a three-hour incubation period, photographs were taken.

[0144] The results are shown in Figure 12. Aggregation of BPTI was observed in the samples to which GSSG was added. On the other hand, aggregation of BPTI was not observed in the samples to which CTAB and the compound (C16N-SS) were added. These results confirm that the combination of the compound (C16N-SS) and CTAB is effective in suppressing BPTI aggregation.

[0145] [Synthesis Example of Compound 2] <Synthesis Example of Compound (C8N-SS)> The synthesis scheme for compound (C8N-SS) is shown below. Compound (C8N-SS) was synthesized according to the following synthesis scheme, with reference to a previously reported study (VMJ Saily et al., Langmuir 2006, 22, 956).

[0146]

[0147] (Synthesis of Compound 4) Compound 2 (1.54 g, 5.47 mmol) was dissolved in anhydrous methanol (16.0 mL), and then triethylamine (1.7953 g, 17.7 mmol) was slowly added dropwise. After stirring at room temperature under a nitrogen atmosphere for 30 minutes, the reaction solution was dried under reduced pressure to obtain Compound 4.

[0148] (Synthesis of compound (C8N-SS)) Compound 4 obtained was dissolved in 2-propanol (11.2 mL), and 1-iodooctane (5.82 g, 24.2 mmol) was slowly added dropwise. After stirring at room temperature under a nitrogen atmosphere for two weeks, the resulting suspension was filtered by suction. The filtrate was dried under reduced pressure, and the resulting residue was purified by silica gel chromatography (NH silica, developing solvent: chloroform / methanol = 9 / 1) and recrystallization with ethanol to obtain C8N-SS. The obtained compound (C8N-SS) is, １ H NMR measurement (400MHz, CDCl ３ , 295K; Figure 13), １３ It was identified by 13C NMR measurement (400 MHz, CDCl3, 298 K; Figure 14) and MALDI-TOF mass spectrometry (measurement mode: Reflector Positive, matrix: 2,5-dihydroxybenzoic acid; Figures 15 and 16).

[0149] [Experimental Example 2] <Protein Folding Test> BPTI was used as the model protein. Reduced-denatured BPTI was prepared using the same method as in Experimental Example 1.

[0150] (Folding test using CTAB and compound (C8N-SS)) Urea and reduced-modified BPTI dissolved in Tris-HCl buffer were added to Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) containing CTAB, C8N-SS, and DMSO. The final concentrations of each component were CTAB 50 mM, compound (C8N-SS) 1.2 mM, DMSO 3% (w / w), urea 246 mM, and reduced-modified BPTI 400 μM. This was incubated at 30°C. Reaction solutions (25.2 μL) taken at predetermined times (0 min, 10 min, 30 min, 60 min, 120 min, 180 min) were mixed with 1 M hydrochloric acid (374.8 μL) and analyzed by reverse-phase HPLC. Flow rate 1.0mL / min －１The process was tracked at a wavelength of 229 nm, and a linear gradient was applied to the solvent ratio during measurement (solvent A: water containing 0.05% TFA, solvent B: acetonitrile containing 0.05% TFA; concentration of solvent A: 95% at 0 minutes, 80% at 15 minutes, 65% at 45 minutes).

[0151] The results are shown in Figure 17. It was confirmed that, over time, the reduced BPTI folded and the natural structure BPTI was formed. The formation rate of the natural structure BPTI after 180 minutes was 7%.

[0152] (Comparison of changes over time in the formation rate of natural structure BPTI) Folding tests of reduced-modified BPTI were performed using the same method as above, except that compound (C16N-SS) and compound (MeN-SS) were used instead of compound (C8N-SS). Furthermore, folding tests of reduced-modified BPTI were performed using the same method as above, except that GSSG was used instead of compound (C8N-SSMeN-SS) and CTAB was not added.

[0153] Figure 18 shows the results of comparing the time-dependent changes in the formation rate of the native structure BPTI for each compound. Compounds (C16N-SS), (MeN-SS), and (C8N-SS) all showed a faster rate of native structure BPTI formation and a larger proportion of native structure BPTI formation compared to GSSG. Compound (C16N-SS) showed the fastest rate of native structure BPTI formation and the largest proportion of native structure BPTI formation.

[0154] [Experimental Example 3] As a model protein, hirudin, which has three disulfide bonds in its molecule, was used.

[0155] (Preparation of reduced hirudin) Reduced hirudin was prepared by following the method described in M. Iwaoka et al., Chem. Eur. J. 2011, 17, 481.

[0156] (Folding test using CTAB and compound (C16N-SS)) Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) containing CTAB, compound (C16N-SS), and 3% (w / w) DMSO was to be mixed with urea and reduced hirudin dissolved in Tris-HCl buffer. The final concentrations of each component were CTAB 50 mM, compound (C16N-SS) 1.2 mM, DMSO 3% (w / w), urea 2.0 mM, and reduced hirudin 400 μM. This mixture was incubated at 30°C. The reaction solution (4 μL) collected at predetermined times (0 hours, 2 hours, 4 hours, 6 hours, 24 hours, 48 ​​hours, 72 hours) was mixed with 0.1% TFA-containing water (1046 μL) and analyzed by reverse-phase HPLC. Flow rate: 1.2 mL / min －１ The reaction was tracked at a wavelength of 280 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: 0.1% TFA-containing water, solvent B: 0.1% TFA-containing acetonitrile; solvent A content: 95% at 0 min, 77% at 15 min, 72% at 40 min, 15% at 41 min). 0.1% TFA-containing water was added to stop the folding reaction.

[0157] The results are shown in Figure 19. It was confirmed that, over time, the reduced-modified hirudin folded and the native structure hirudin was formed. The formation rate of the native structure hirudin was 94% after 72 hours.

[0158] (Folding test using CTAB and compound (C8N-SS)) Urea and reduced hirudin dissolved in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) containing CTAB, compound (C8N-SS), and DMSO were added. The final concentrations of each component were CTAB 50 mM, compound (C8N-SS) 1.2 mM, DMSO 3% (w / w), urea 2.0 mM, and reduced hirudin 400 μM. This was incubated at 30°C. The reaction solution (4 μL) sampled at predetermined times (0 hours, 2 hours, 4 hours, 6 hours, 24 hours, 48 ​​hours, 72 hours) was mixed with 0.1% TFA-containing water (1046 μL) and analyzed by reverse-phase HPLC. Flow rate 1.2mL / min －１ The reaction was tracked at a wavelength of 280 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: 0.1% TFA-containing water, solvent B: 0.1% TFA-containing acetonitrile; solvent A content: 95% at 0 min, 77% at 15 min, 72% at 40 min, 15% at 41 min). 0.1% TFA-containing water was added to stop the folding reaction.

[0159] The results are shown in Figure 20. It was confirmed that, over time, the reduced-modified hirudin folded and the native structure hirudin was formed. The formation rate of the native structure hirudin was 78% after 72 hours.

[0160] (Folding test using CTAB and compound (MeN-SS)) Urea and reduced hirudin dissolved in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) containing CTAB, compound (MeN-SS), and DMSO were added. The final concentrations of each component were CTAB 50 mM, compound (MeN-SS) 1.2 mM, DMSO 3% (w / w), urea 2.0 mM, and reduced hirudin 400 μM. This was incubated at 30°C. The reaction solution (4 μL) sampled at predetermined times (0 hours, 2 hours, 4 hours, 6 hours, 24 hours, 48 ​​hours, 72 hours) was mixed with 0.1% TFA-containing water (1046 μL) and analyzed by reverse-phase HPLC. Flow rate 1.2mL / min －１ The reaction was tracked at a wavelength of 280 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: 0.1% TFA-containing water, solvent B: 0.1% TFA-containing acetonitrile; solvent A content: 95% at 0 min, 77% at 15 min, 72% at 40 min, 15% at 41 min). 0.1% TFA-containing water was added to stop the folding reaction.

[0161] The results are shown in Figure 21. It was confirmed that, over time, the reduced-modified hirudin folded and the native structure hirudin was formed. The formation rate of the native structure hirudin after 72 hours was 21%.

[0162] (Folding test using compound (MeN-SS)) To a Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) containing compound (MeN-SS) and DMSO, urea and reduced-modified hirudin dissolved in Tris-HCl buffer were added. The final concentrations of each component were 1.2 mM compound (MeN-SS), 3% (w / w) DMSO, 2.0 mM urea, and 400 μM reduced-modified hirudin. This was incubated at 30°C. At predetermined times (0 hours, 2 hours, 4 hours, 6 hours, 24 hours, 48 ​​hours, 72 hours), 4 μL of the reaction solution was sampled and mixed with 1046 μL of 0.1% TFA-containing water, and analyzed by reverse-phase HPLC. Flow rate: 1.2 mL / min－１ The reaction was tracked at a wavelength of 280 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: 0.1% TFA-containing water, solvent B: 0.1% TFA-containing acetonitrile; solvent A content: 95% at 0 min, 77% at 15 min, 72% at 40 min, 15% at 41 min). 0.1% TFA-containing water was added to stop the folding reaction.

[0163] The results are shown in Figure 22. It was confirmed that, over time, the reduced-modified hirudin folded and the native structure hirudin was formed. The formation rate of the native structure hirudin after 72 hours was 23%.

[0164] (Folding test using GSSG) Urea and reduced hirudin dissolved in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) containing GSSG and DMSO were added. The final concentrations of each component were GSSG 1.2 mM, DMSO 3% (w / w), urea 2.0 mM, and reduced hirudin 400 μM. This was incubated at 30°C. The reaction solution (4 μL) collected at predetermined times (0 hours, 2 hours, 4 hours, 6 hours, 24 hours, 48 ​​hours, 72 hours) was mixed with 0.1% TFA-containing water (1046 μL) and analyzed by reverse-phase HPLC. Flow rate 1.2 mL / min －１ The reaction was tracked at a wavelength of 280 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: 0.1% TFA-containing water, solvent B: 0.1% TFA-containing acetonitrile; solvent A content: 95% at 0 min, 77% at 15 min, 72% at 40 min, 15% at 41 min). 0.1% TFA-containing water was added to stop the folding reaction.

[0165] The results are shown in Figure 23. It was confirmed that, over time, the reduced-modified hirudin folded and the native structure hirudin was formed. The formation rate of the native structure hirudin after 72 hours was 6%.

[0166] (Comparison of changes over time in the rate of formation of natural structural hirudin) Based on the results in Figures 19-23, Figure 24 shows the results of comparing the changes over time in the rate of formation of natural structural hirudin for each compound. Compounds (C16N-SS), (MeN-SS), and (C8N-SS) all showed a faster rate of formation of natural structural hirudin and a larger rate of formation of natural structural hirudin compared to GSSG. The rate and rate of formation of natural structural hirudin were highest for compound (C16N-SS), followed by compound (C8N-SS).

[0167] [Experimental Example 4] Endothelin-1 (ET-1), which has two disulfide bonds in its molecule, was used as a model protein.

[0168] <Preparation of Reduced-Modified ET-1> (Synthesis of ET-1) Biotage® Initiator + Alstra ＴＭ A solid-phase synthesis tube (ISOLUTE SPE ACCESSORIES, 10 ml Reactor Vial PTFE) containing Fmoc-Trp(Boc)-Wang Resin (278 mg, 0.15 mmol) was placed in a Biotage Japan container. N,N-dimethylformamide (DMF) (4.50 mL) was added and the mixture was swollen at 70°C for 20 minutes, after which the reaction solution was removed. Piperidine (20% in DMF, 4.50 mL) was added and the mixture was shaken at room temperature for 3 minutes, after which the reaction solution was removed. Piperidine (20% in DMF, 4.50 mL) was added and the mixture was shaken at room temperature for 10 minutes, after which the reaction solution was removed. The mixture was washed four times with DMF (4.50 mL) and the reaction proceeded to the next step. N-terminal amino acid (0.15 mmol), HBTU (0.6 M in DMF) (0.147 mmol), HOBt·H ２0.147 mmol of 0.5 M in DMF and 0.30 mmol of N,N-diisopropylethylamine (DIEA) (2 M in NMP) were added to the resin and shaken at 50°C for 60 minutes, after which the reaction solution was removed. The mixture was washed four times with DMF (4.50 mL), and thereafter the above procedure was repeated according to the amino acid sequence to extend the peptide chain. Fmoc-Cys-(Trt)-OH and Fmoc-His-(Trt)-OH were shaken at room temperature for 60 minutes to prevent racemization. Fmoc-Ile-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, and Fmoc-Ser(tBu)-OH were used for peptide elongation. At the end of peptide chain elongation, the peptides were washed six times with dichloromethane (DCM) (4.5 mL) and then dried for 2 hours.

[0169] (Excision of ET-1) The peptide was excised from the resin and freeze-dried using the following procedure. The resin was washed three times with methanol (4.5 mL) and dried in a desiccator. The deprotection cocktail was added to the resin, and the mixture was gently shaken every 30 minutes at room temperature and allowed to stand for 90 minutes. The deprotection cocktail was prepared by pre-mixing 4000 μL of trifluoroacetic acid (TFA), 250 μL of water, 250 μL of phenol, 250 μL of thioanisole (TA), 125 μL of triisopropylsilane (TIS), and 125 μL of 1,2-ethanedithiol (EDT). The filtrate was collected in a 15 mL centrifuge tube. 500 μL of TFA was added to the synthesis tube, and the filtrate was collected in the centrifuge tube. This procedure was repeated three times. 35 mL of diethyl ether was added to the centrifuge tube from which the filtrate was collected, and the mixture was thoroughly mixed. The sample was centrifuged (4°C, 3500 × g, 5 min) and the supernatant was removed. This procedure was repeated three times. After standing in a fume hood for 10 minutes to dry, it was dried in a desiccator for at least 2 hours. The dried sample was dispersed in deionized water and freeze-dried.

[0170] (Preparation of reduced-modified ET-1) To the molar amount of crudely purified ET-1, 5 to 10 equivalents of DTT and 0.1% TFA-containing water containing 8M urea were added, and 0.1% TFA-containing acetonitrile was added until the solution became clear. At this time, the sample was prepared so that the ratio of the added solvent (0.1% TFA-containing water):(0.1% TFA-containing acetonitrile) was greater than 70 / 30. The solution was passed through a membrane (Membrane Solutions) (hydrophobic, PVDF φ25 mm / φ0.45 μm) and purified by reverse-phase HPLC. HPLC purification was performed using a PU-4186 pump and UV-2075PLUS detector manufactured by JASCO Corporation (Tokyo, Japan), and an Inertsil ODS-HL column (10 mm x 250 mm) manufactured by GL Sciences Co., Ltd. (Tokyo, Japan), at a flow rate of 4.7 mL / min. －１ The procedure was carried out as follows. The recovered fraction was freeze-dried, and the resulting powder was stored at -30°C.

[0171] (Reagents) ISOLUTE SPE ACCESSORIES, 10 ml. Reactor Vial PTFE was purchased from Biotage Japan Co., Ltd. (Tokyo, Japan). Fmoc-Trp(Boc)-Wang Resin, HBTU, HOBt-H ２O, Fmoc-Ile-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, and Fmoc-Ser(tBu)-OH were purchased from Watanabe Chemical Co., Ltd. (Hiroshima, Japan). DMF, piperidine, NMP, TFA, and diethyl ether were purchased from Kishida Chemical Co., Ltd. (Osaka, Japan). DIEA was purchased from Nacalai Tesque Co., Ltd. (Kyoto, Japan). TIS, thioanisole, and 1,2-ethanedithiol were purchased from Tokyo Kogyo Kasei Co., Ltd. (Tokyo, Japan). DCM was purchased from AGC Inc. (Chiba, Japan). Methanol was purchased from Godo Co., Ltd. (Tokyo, Japan). Phenol was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

[0172] (Equipment) HPLC purification was performed using a PU-4186 pump and UV-2075PLUS detector manufactured by JASCO Corporation (Tokyo, Japan), and an Inertsil ODS-HL column (10 mm x 250 mm) manufactured by GL Sciences Co., Ltd. (Tokyo, Japan).

[0173] (Folding test using CTAB and compound (C16N-SS)) Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) containing CTAB, compound (C16N-SS), and DMSO was to be mixed with urea dissolved in Tris-HCl buffer and reduced-modified ET-1. The final concentrations of each component were CTAB 50 mM, compound (C16N-SS) 2 mM, DMSO 5% (w / w), urea 0.5 mM, and reduced-modified ET-1 1000 μM. This mixture was incubated at 30°C. The reaction solution (1.6 μL) collected at predetermined times (0 min, 10 min, 30 min, 60 min, 120 min, 180 min) was mixed with 0.1% TFA-containing water (1048.4 μL) and analyzed by reverse-phase HPLC. Flow rate: 1.0 mL / min －１ The reaction was tracked at a wavelength of 280 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: 0.1% TFA-containing water, solvent B: 0.1% TFA-containing acetonitrile; concentration of solvent A: 70% at 0 min, 50% at 20 min, 5% at 22 min). 0.1% TFA-containing water was added to stop the folding reaction.

[0174] The results are shown in Figure 25. It was confirmed that, over time, the reduced-modified ET-1 folded, and the natural structure ET-1 was formed. At 180 minutes, the formation rate of the natural structure hirudin was 63%.

[0175] (Folding test using GSSG) Urea and reduced-modified ET-1 dissolved in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) containing GSSG and DMSO were added. The final concentrations of each component were GSSG 2 mM, DMSO 5% (w / w), urea 0.5 mM, and reduced-modified ET-1 1000 μM. This was incubated at 30°C. Reaction solutions (1.6 μL) taken at predetermined times (0 min, 10 min, 30 min, 60 min, 120 min, 180 min) were mixed with 0.1% TFA-containing water (1048.4 μL) and analyzed by reverse-phase HPLC. Flow rate 1.0 mL / min －１The reaction was tracked at a wavelength of 280 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: 0.1% TFA-containing water, solvent B: 0.1% TFA-containing acetonitrile; concentration of solvent A: 70% at 0 min, 50% at 20 min, 5% at 22 min). 0.1% TFA-containing water was added to stop the folding reaction.

[0176] The results are shown in Figure 26. It was confirmed that, over time, the reduced-modified ET-1 folded, and the natural structure ET-1 was formed. The formation rate of the natural structure ET-1 after 180 minutes was 7%.

[0177] (Comparison of changes over time in the formation rate of natural structure ET-1) Based on the results in Figures 25 and 26, Figure 27 shows the results of comparing the changes over time in the formation rate of natural structure ET-1 for each compound. Compared to GSSG, compound (C16N-SS) showed a faster rate of formation of natural structure ET-1 and a larger proportion of natural structure ET-1 formation.

[0178] (Evaluation of the protein aggregation inhibitory effect of compound (C16N-SS)) Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) containing CTAB, compound (C16N-SS), and DMSO was to be mixed with urea dissolved in Tris-HCl buffer and reduced-denatured ET-1. The final concentrations of each component were CTAB 50 mM, compound (C16N-SS) 2 mM, DMSO 5% (w / w), urea 0.5 mM, and reduced-denatured ET-1 1000 μM. This mixture was incubated at 30°C. After incubation for 180 minutes, photographs were taken. In addition, the incubation was performed at 30°C using the same method as above, except that oxidized glutathione (GSSG) was used instead of compound (C16N-SS) and CTAB was not used. The images were photographed after 180 minutes of incubation.

[0179] The results are shown in Figure 28. Aggregation of ET-1 was observed in the sample to which GSSG was added. On the other hand, aggregation of ET-1 was not observed in the sample to which the compound (C16N-SS) and CTAB were added. These results confirm that the combination of the compound (C16N-SS) and CTAB is effective in suppressing the aggregation of ET-1.

[0180] The present invention provides a protein folding agent capable of promoting protein folding at protein concentrations of several hundred μM or more, a protein folding composition containing the protein folding agent, a protein folding kit, and a protein folding method using the protein folding agent.

[0181] While preferred embodiments of the present invention have been described above, the present invention is not limited to these embodiments. Additions, omissions, substitutions, and other modifications are possible without departing from the spirit of the invention. The present invention is not limited by the foregoing description, but only by the scope of the appended claims.

Claims

1. A protein folding agent comprising compound (A) represented by the following general formula (A). [In the formula, R １ ~R ６ Each of these independently represents an aliphatic hydrocarbon group having 1 to 24 carbon atoms. １ and Y ２ Each of these independently represents an aliphatic hydrocarbon group having 1 to 10 carbon atoms, which may contain an oxygen atom. n is an integer greater than or equal to 1, and X ｎ－ This represents an n-valent anion.

2. The protein folding agent according to claim 1, wherein the compound (A) is a compound represented by the following general formula (A1). [In the formula, R １ ~R ６ Each of these independently represents an aliphatic hydrocarbon group having 1 to 24 carbon atoms. n is an integer greater than or equal to 1, and X ｎ－ This represents an n-valent anion.

3. The protein folding agent according to claim 2, wherein the compound (A) is a compound represented by the following general formula (A1-1). [In the formula, R １ and R ４ each independently represents an aliphatic hydrocarbon group having 1 to 24 carbon atoms. n is an integer of 1 or more, and X ｎ－ represents an n-valent anion. ] 4. The protein folding agent according to claim 3, wherein the compound (A) is a compound represented by the following general formula (A1-1-1). [In the formula, m1 and m2 each independently represent integers from 1 to 24. n is an integer greater than or equal to 1, and X ｎ－ This represents an n-valent anion.

5. The aforementioned X ｎ－ A protein folding agent according to any one of claims 1 to 4, wherein is a halide ion.

6. A protein folding composition comprising a protein folding agent according to any one of claims 1 to 4 and a protein aggregation inhibitor.

7. The protein folding composition according to claim 6, wherein the protein aggregation inhibitor is a surfactant.

8. A protein folding kit comprising a protein folding agent according to any one of claims 1 to 4.

9. The protein folding kit according to claim 8, further comprising a protein aggregation inhibitor.

10. The protein folding kit according to claim 8, further comprising a polar organic solvent.

11. A method for folding a protein, comprising step (a) of incubating a protein in the presence of a protein folding agent according to any one of claims 1 to 4.

12. The protein folding method according to claim 11, wherein step (a) is a step of incubating the protein in the presence of the protein folding agent and the protein aggregation inhibitor.

13. The method for folding a protein according to claim 11, further comprising the step (b) of adding a polar organic solvent to the folding reaction solution after step (a).

14. The protein folding method according to claim 11, wherein the protein is an unfolding protein or a misfolding protein.

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