Peptide, droplet forming kit, droplet forming method, hydrophobic substance concentration method, and protein folding method

Abstract

The present invention pertains to a peptide having the following features (a)-(c): (a) 2 g or more of the peptide is dissolved at 20°C in 100 mL of a 50 mM Tris-hydrochloric acid buffer (pH 7.5) containing 0.3 M sodium chloride; (b) the peptide undergoes a reversible phase transition in the Tris-hydrochloric acid buffer, and has a transition temperature of 40-70°C when 2 g is dissolved in 100 mL of the Tris-hydrochloric acid buffer; and (c) when 2 g of the peptide is dissolved in 100 mL of the Tris-hydrochloric acid buffer, a droplet is formed under temperature conditions equal to or greater than the transition temperature.

Description

Peptide, droplet formation kit, droplet formation method, method for concentrating hydrophobic substances, and protein folding method

[0001] The present invention relates to a peptide, a droplet formation kit, a droplet formation method, a method for concentrating a hydrophobic substance, and a protein folding method.This application claims priority to Japanese Patent Application No. 2023-137002 filed in Japan on August 25, 2023, and Japanese Patent Application No. 2024-078820 filed in Japan on May 14, 2024, the contents of which are incorporated herein by reference.

[0002] Aqueous two-phase systems (liquid-liquid phase separation), in which an aqueous solution spontaneously separates into two phases, are a useful phenomenon for concentrating and separating solutes. Polymers such as polyethylene glycol are well known as materials that form aqueous two-phase systems. However, polymers have high molecular weights, which increases the viscosity of aqueous solutions. Furthermore, problems arise, such as sample heterogeneity and lot-to-lot differences due to the molecular weight distribution of the polymer.

[0003] Elastin-like peptides (ELPs) are known as polypeptides that form aqueous two-phase systems at high temperatures (Non-Patent Document 1). ELPs include (VPGXG) ｎ (X is an amino acid residue other than proline, and n is an integer of 1 or greater). ELPs with n > 40 are commonly used. Reported uses of ELPs include the recovery of heavy metal ions by forming an aqueous two-phase system (Non-Patent Document 2), separation of proteins with an ELPs sequence introduced at their termini by forming an aqueous two-phase system (Non-Patent Document 3), and use in drug delivery systems by covalently binding small drug molecules such as anticancer drugs to the termini of ELPs (Non-Patent Document 4). Furthermore, based on the ELPs sequence, short-chain peptides made only from natural amino acids that form aqueous two-phase systems are being developed (Non-Patent Documents 1, 5, 6).

[0004] Shogo Sumiyoshi, et al. Development of truncated elastin-like peptide analogues with improved temperature-response and self-assembling properties. Scientific Reports, 2022, 12, 19414.Shogo Sumiyoshi, et al. Metal ion scavenging activity of elastin-like peptide analogues containing a cadmium ion binding sequence. Scientific Reports, 2022, 12, 1861.Agnes Yeboah, et al. Elastin-like polypeptides: A strategic fusion partner for biologics. Biotechnology and Bioengineering, 2016, 113, 1617.Jose Carlos Rodriguez-Cabello, et al. Elastin-like polypeptides in drug delivery. Advanced Drug Delivery Reviews, 2016, 97, 85.Woo-jin Jeong, et al. Modular Self-Assembling Peptide Platform with a Tunable Thermoresponsiveness via a Single Amino Acid Substitution. Advanced Functional Materials, 2018, 28, 1803114.Suguru Taniguchi, et al. Development of short and highly potent self-assembling elastin-derived pentapeptide repeats containing aromatic amino acid residues. Journal of Peptide Science, 2016, 22, 36.

[0005] High molecular weight polymers such as polyethylene glycol, which have traditionally been used to form aqueous two-phase systems, have problems such as high viscosity in aqueous solutions and non-uniform molecular weight distribution. If low molecular weight materials could be used instead of high molecular weight polymers to form aqueous two-phase systems, the problems associated with the use of high molecular weight polymers could be eliminated. Short-chain peptides that form aqueous two-phase systems based on ELP sequences have been developed, but they have low water solubility, making it difficult to prepare aqueous solutions with sufficient concentrations for droplet formation. Furthermore, no methods are known that can concentrate and recover proteins using aqueous two-phase systems.

[0006] Furthermore, peptides that can promote the folding of proteins into their native structures are expected to be useful in the production of protein pharmaceuticals.

[0007] Therefore, the present invention provides peptides that can be used for protein concentration and recovery or protein folding, droplet formation kits containing the peptides, and droplet formation methods, hydrophobic substance concentration methods, and protein folding methods using the peptides.

[0008] The present invention includes the following aspects. [1] A peptide having the following characteristics (a) to (c): (a) 2 g or more of the peptide dissolves in 100 mL of 50 mM Tris-HCl buffer (pH 7.5) containing 0.3 M sodium chloride at 20°C; (b) the peptide undergoes reversible phase transition in the Tris-HCl buffer, and the transition temperature when 2 g of the peptide is dissolved in 100 mL of the Tris-HCl buffer is 40 to 70°C; and (c) the peptide forms droplets at a temperature equal to or higher than the transition temperature when 2 g of the peptide is dissolved in 100 mL of the Tris-HCl buffer. [2] The peptide according to [1], which contains the amino acid sequence set forth in SEQ ID NO: 1: VPGXG (SEQ ID NO: 1) [wherein V represents a valine residue, P represents a proline residue, G represents a glycine residue, and X represents any amino acid residue other than a proline residue]. [3] The peptide according to [2], which contains, in addition to the amino acid sequence set forth in SEQ ID NO: 1, one or more hydrophobic aliphatic amino acid residues at at least one of the N-terminus and the C-terminus. [4] The peptide according to [3], wherein the hydrophobic aliphatic amino acid residue is at least one selected from the group consisting of leucine residue, isoleucine residue, valine residue, alanine residue, and methionine residue. [5] The peptide according to [3], comprising an amino acid sequence represented by the following general formula (I): (Z1): ｎ１ - (VPGXG) ｍ -(Z2) ｎ２ (I) [In the formula, Z1 and Z2 each independently represent a hydrophobic aliphatic amino acid residue. V represents a valine residue, P represents a proline residue, G represents a glycine residue, and X represents any amino acid residue other than a proline residue. n1 and n2 each independently represent an integer of 0 to 3, and n1 + n2 ≧ 1. m represents an integer of 3 to 5.] [6] The peptide according to [5], wherein the amino acid sequence represented by the general formula (I) is the amino acid sequence set forth in SEQ ID NO: 2. LL-(VPGXG) ４(SEQ ID NO: 2) [In the formula, L represents a leucine residue, V represents a valine residue, P represents a proline residue, G represents a glycine residue, and X represents any amino acid residue other than a proline residue.] [7] The peptide according to any one of [1] to [6], which is used to concentrate a hydrophobic substance. [8] The peptide according to [7], wherein the hydrophobic substance is a denatured protein. [9] The peptide according to any one of [1] to [8], which contains at least one cysteine ​​residue.

[10] The peptide according to [9], which contains an amino acid sequence represented by the following general formula (II): (Z1) ｎ１ -(Cx) ｐ１ - (VPGXG) ｍ1 -(Cy) ｐ２ - (VPGXG) ｍ２ -(Cx) ｐ３ -(Z2) ｎ２ (II) [Wherein, Z1 and Z2 each independently represent a hydrophobic aliphatic amino acid residue. V represents a valine residue, P represents a proline residue, G represents a glycine residue, and X represents any amino acid residue other than a proline residue. Cx, Cy, and Cz each independently represent 1 to 5 amino acid residues including at least one cysteine ​​residue. n1 and n2 each independently represent an integer of 0 to 3, and n1 + n2 ≧ 1. m1 and m2 each independently represent an integer of 0 to 5, and 3 ≦ m1 + m2 ≦ 5. p1, p2, and p3 each independently represent 0 or 1, and p1 + p2 + p3 ≧ 1.]

[11] The peptide according to

[10] , wherein the amino acid sequence represented by the general formula (II) is the amino acid sequence set forth in SEQ ID NO: 4. LL-(VPGXG) ２ -CGHC-(VPGXG) ２(SEQ ID NO: 4) [In the formula, L represents a leucine residue, V represents a valine residue, P represents a proline residue, G represents a glycine residue, C represents a cysteine ​​residue, H represents a histidine residue, and X represents any amino acid residue other than a proline residue.]

[12] A peptide according to any one of [1] to

[11] , which is used for protein folding.

[13] A droplet formation kit comprising the peptide according to any one of [1] to

[12] .

[14] A droplet formation method comprising: (A) preparing an aqueous solution containing the peptide according to any one of [1] to [6] or [9] to

[11] ; and (B) incubating the aqueous solution at a temperature higher than the transition temperature of the peptide.

[15] A method for concentrating a hydrophobic substance, comprising: (A1) preparing an aqueous solution containing the peptide according to any one of [1] to [6] or [9] to

[11] and a hydrophobic substance; and (B1) incubating the aqueous solution at a temperature higher than the transition temperature of the peptide.

[16] The method for concentrating a hydrophobic substance according to

[15] , wherein the hydrophobic substance is a denatured protein.

[17] A method for folding a protein, comprising: (A2) preparing an aqueous solution containing the peptide according to any one of [9] to

[11] and a protein; and (B2) incubating the aqueous solution.

[18] The method for folding a protein according to

[17] , wherein the protein is a denatured protein.

[0009] According to the present invention, there are provided peptides that can be used for protein concentration and recovery or protein folding, droplet formation kits containing the peptides, and droplet formation methods, hydrophobic substance concentration methods, and protein folding methods using the peptides.

[0010] 1 shows an RP-HPLC chart of LL-ELP4 synthesized in Experimental Example 1. 1 shows a MALDI-TOF-MS spectrum of LL-ELP4 synthesized in Experimental Example 1. 1 shows the results of monitoring the temperature-dependent change in transmittance in a 1% by mass solution of FF-ELP4 (a) and a 2% by mass solution of LL-ELP4 (b) prepared in Experimental Example 1. 1 shows photographs showing the state of the solution below the transition temperature and above the transition temperature. 1 shows the results of monitoring the temperature-dependent change in transmittance in a 5% by mass solution of LL-EPL4 prepared in Experimental Example 1. 1 shows phase-contrast microscope images at 60°C of a 1% by mass solution of FF-ELP4 (a) and a 2% by mass solution of LL-EPL4 (b) prepared in Experimental Example 1. 1 shows an RP-HPLC chart of LL-ELP4-SS synthesized in Experimental Example 2. 1 shows a MALDI-TOF-MS spectrum of LL-ELP4 synthesized in Experimental Example 2. 1 shows a phase-contrast microscope image at 60°C of a mixed peptide solution of LL-ELP4 (2% by mass) and LL-ELP4-SS (0.16% by mass) prepared in Experimental Example 2. 1 shows the results of a folding test of reduced-modified BPTI in a mixed peptide solution of LL-ELP4 (2% by mass) and LL-ELP4-SS (0.16% by mass) prepared in Experimental Example 2. 1 shows a phase-contrast microscope image at 60°C of a 2% by mass solution of LL-ELP4-SS prepared in Experimental Example 2. 1 shows the results of a folding test of reduced-modified BPTI in a 2% by mass solution of LL-ELP4-SS prepared in Experimental Example 2.

[0011] A numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0012] The term "comprise" means that it may contain components other than the target component. The term "consist of" means that it does not contain components other than the target component. The term "consist essentially of" means that it does not contain components other than the target component in a manner that would exert a special function (such as a manner that would completely lose the effects of the invention). In this specification, when "comprise" is used, it includes both "consist of" and "consist essentially of" embodiments.

[0013] "Peptide" refers to a polymer of amino acids linked by amide bonds. Peptides are described using the commonly accepted one-letter or three-letter codes unless otherwise specified. The amino acid sequence of a peptide is described from the N-terminus to the C-terminus unless otherwise specified. A peptide may be a polymer of natural amino acids, a polymer of natural amino acids and unnatural amino acids, or a polymer of unnatural amino acids. From the viewpoints of biocompatibility and biodegradability, a peptide is preferably a polymer of natural amino acids and preferably does not contain unnatural amino acid residues.

[0014] "Natural amino acids" are amino acids that occur in nature, and are preferably selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0015] "Unnatural amino acids" are amino acids that do not occur in nature. Unnatural amino acids include, for example, modified derivatives of natural amino acids.

[0016] <Peptide> A first aspect of the present invention is a peptide (hereinafter also referred to as "peptide (P)") having the following characteristics (a) to (c): (a) 2 g or more of the peptide dissolves in 100 mL of 50 mM Tris-HCl buffer (pH 7.5) containing 0.3 M sodium chloride at 20°C; (b) the peptide undergoes reversible phase transition in the Tris-HCl buffer, and the transition temperature when 2 g of the peptide is dissolved in 100 mL of the Tris-HCl buffer is 40 to 70°C; and (c) the peptide forms droplets at temperatures equal to or higher than the transition temperature when 2 g of the peptide is dissolved in 100 mL of the Tris-HCl buffer.

[0017] In one embodiment, the peptide (P) is used to form droplets in an aqueous two-phase system. In an aqueous two-phase system, an aqueous solution spontaneously separates into two phases. In one embodiment, an aqueous solution containing the peptide (P) is dissolved in the peptide (P), and the aqueous solution separates into two phases due to aggregation of the peptide (P), forming droplets.

[0018] By virtue of the above characteristics (a) to (c), peptide (P) can form droplets of appropriate size under relatively mild conditions without excessively increasing the viscosity of its aqueous solution. Furthermore, hydrophobic substances (e.g., denatured proteins) can be concentrated within the droplets. Furthermore, peptides (P) containing cysteine ​​residues can promote protein folding.

[0019] (Regarding (a)) The 50 mM Tris-HCl buffer (pH 7.5) containing 0.3 M sodium chloride in (a) above (hereinafter also referred to as "NaCl-containing Tris-HCl (pH 7.5)") can be prepared by dissolving sodium chloride in 50 mM Tris-HCl buffer (pH 7.5) to a final concentration of 0.3 M. The 50 mM Tris-HCl buffer (pH 7.5) can be prepared, for example, by dissolving 0.6057 g of trishydroxymethylaminomethane in 50 to 80 mL of water (distilled water, etc.), adjusting the pH to 7.5 with hydrochloric acid, and then adding water to bring the total volume to 100 mL.

[0020] At 20°C, 2 g or more of peptide (P) dissolves in 100 mL of NaCl-containing Tris-HCl (pH 7.5). From the viewpoint of forming droplets under relatively mild conditions, it is preferable that 20 g or more of peptide (P) does not dissolve in 100 mL of NaCl-containing Tris-HCl (pH 7.5) at 20°C. The maximum dissolution amount of peptide (P) in 100 mL of NaCl-containing Tris-HCl (pH 7.5) at 20°C is preferably 3 g or more, more preferably 5 g or more. The maximum dissolution amount is preferably 20 g or less, more preferably 15 g or less. The maximum dissolution amount of the peptide of this embodiment in 100 mL of NaCl-containing Tris-HCl (pH 7.5) at 20°C is preferably 2 to 15 g.

[0021] The maximum dissolution amount of peptide (P) in 100 mL of NaCl-containing Tris-HCl (pH 7.5) at 20°C can be confirmed, for example, as follows. 100 mL of NaCl-containing Tris-HCl (pH 7.5) is placed in a container (beaker, flask, etc.), and peptide (P) is added while maintaining the temperature at 20°C using a water bath, ultrasonic cleaner, etc. The addition of peptide (P) is preferably carried out while stirring the NaCl-containing Tris-HCl (pH 7.5) with a stirrer, etc. The peptide (P) is added little by little to dissolve, and once it has been confirmed that the entire amount of added peptide (P) has dissolved, further peptide (P) is added. The amount of peptide (P) added up to the point just before the newly added peptide (P) no longer dissolves in 100 mL of NaCl-containing Tris-HCl (pH 7.5) can be determined as the maximum dissolution amount of peptide (P).

[0022] (Regarding (b)) The Tris-HCl buffer in (b) is the same as the NaCl-containing Tris-HCl (pH 7.5) in (a). Peptide (P) undergoes a reversible phase transition in a peptide solution prepared by dissolving it in NaCl-containing Tris-HCl (pH 7.5). "Phase transition" refers to a change from a state without liquid-liquid phase separation to a state with liquid-liquid phase separation, or vice versa. "Transition temperature" refers to the temperature at which a phase transition occurs. A peptide solution prepared by dissolving 2 g of peptide (P) in 100 mL of NaCl-containing Tris-HCl (pH 7.5) (hereinafter also referred to as "peptide (P) solution") undergoes liquid-liquid phase separation at temperatures above the transition temperature to form an aqueous two-phase system. At temperatures below the transition temperature, the liquid-liquid phase separation disappears, resulting in a homogeneous solution. A peptide (P) solution reversibly changes between an aqueous one-phase system and an aqueous two-phase system at the transition temperature.

[0023] When the peptide (P) solution undergoes liquid-liquid phase separation to form an aqueous two-phase system, droplets are formed in the peptide (P) solution, and the transmittance of the peptide (P) solution decreases. Therefore, the presence or absence of a phase transition can be confirmed by observing the change in the transmittance of the peptide (P) solution. The transmittance of the peptide (P) solution can be confirmed, for example, by measuring the absorbance at 600 nm using a spectrophotometer.

[0024] The transition temperature of the peptide (P) solution is 40 to 70° C. The transition temperature is preferably 45 to 65° C., more preferably 50 to 60° C., and even more preferably 50 to 55° C.

[0025] (Regarding (c)) The Tris-HCl buffer in (c) is the same as the NaCl-containing Tris-HCl (pH 7.5) in (a). In a peptide solution prepared by dissolving peptide (P) in NaCl-containing Tris-HCl (pH 7.5), droplets are formed at a temperature equal to or higher than the transition temperature. The "transition temperature" is the same as the "transition temperature" described in (b) above, and is the temperature at which a phase transition occurs. In a peptide (P) solution prepared by dissolving 2 g of peptide (P) in 100 mL of NaCl-containing Tris-HCl (pH 7.5), liquid-liquid phase separation occurs at a temperature equal to or higher than the transition temperature to form an aqueous two-phase system. When the peptide (P) solution undergoes liquid-liquid phase separation to form an aqueous two-phase system, droplets are formed in the peptide (P) solution. The formation of droplets can be confirmed, for example, by observation with a phase-contrast microscope, measurement of transmittance with a spectrophotometer (e.g., measurement of absorbance at 600 nm), etc.

[0026] (Specific Example of Peptide (P)) A specific example of peptide (P) is a peptide containing the amino acid sequence set forth in SEQ ID NO: 1 below (hereinafter also referred to as "amino acid sequence (1)"): VPGXG (SEQ ID NO: 1) [wherein V represents a valine residue, P represents a proline residue, G represents a glycine residue, and X represents any amino acid residue other than a proline residue.]

[0027] In the amino acid sequence (1), X may be an amino acid residue derived from an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine. Among these, X is preferably an alanine residue, a valine residue, a leucine residue, an isoleucine residue, or a methionine residue, and more preferably a valine residue.

[0028] In addition to the amino acid sequence (1), the peptide (P) preferably contains one or more hydrophobic aliphatic amino acid residues at at least one of the N-terminus and the C-terminus. A "hydrophobic aliphatic amino acid residue" is an amino acid residue derived from a hydrophobic amino acid that does not contain an aromatic ring. Specific examples of the hydrophobic aliphatic amino acid residue include at least one selected from the group consisting of leucine residue, isoleucine residue, valine residue, alanine residue, and methionine residue. Among these, leucine residue, isoleucine residue, or valine residue is preferred from the viewpoint of hydrophobicity, and leucine residue is more preferred.

[0029] The number of hydrophobic aliphatic amino acid residues added to at least one of the N-terminus and the C-terminus is preferably 1 to 5, more preferably 2 to 4, and even more preferably 2 or 3. When the number of amino acid residues added to the N-terminus and / or the C-terminus is within the above-mentioned preferred range, droplets of appropriate size are likely to be formed. When multiple hydrophobic aliphatic amino acid residues are added, the multiple hydrophobic aliphatic amino acid residues may be the same or different. The hydrophobic aliphatic amino acid residues may be added to the N-terminus, the C-terminus, or both the N-terminus and the C-terminus. When hydrophobic aliphatic amino acid residues are added to both the N-terminus and the C-terminus, the type and number of hydrophobic aliphatic amino acid residues added to the N-terminus and the C-terminus may be the same or different. For example, one may be added to each of the N-terminus and the C-terminus, two may be added to the N-terminus and one to the C-terminus, or one may be added to the N-terminus and two to the C-terminus.

[0030] In one embodiment, the peptide (P) is preferably a peptide consisting of an amino acid sequence represented by the following general formula (I) (hereinafter also referred to as "peptide (P-I)"): (Z1) ｎ１ - (VPGXG) ｍ -(Z2) ｎ２(I) [In the formula, Z1 and Z2 each independently represent a hydrophobic aliphatic amino acid residue. V represents a valine residue, P represents a proline residue, G represents a glycine residue, and X represents any amino acid residue other than a proline residue. n1 and n2 each independently represent an integer of 0 to 3, provided that n1 + n2 ≥ 1. m represents an integer of 3 to 5.]

[0031] In the formula (I), examples of the hydrophobic aliphatic amino acid residues in Z1 and Z2 include those similar to those described above. As the hydrophobic aliphatic amino acid residue, a leucine residue, an isoleucine residue, or a valine residue is preferred, and a leucine residue is more preferred.

[0032] In the formula (I), n1 and n2 preferably satisfy the relationship 1≦n1+n2≦4, and more preferably satisfy the relationship 2≦n1+n2≦3. Preferably, n1 is 2 or 3 and n2 is 0, or n1 is 0 and n2 is 2 or 3, more preferably, n1 is 2 or 3 and n2 is 0, and even more preferably, n1 is 2 and n2 is 0.

[0033] When n1 is an integer of 2 or more, two or more Z1 may be the same as or different from each other. When n2 is an integer of 2 or more, two or more Z2 may be the same as or different from each other.

[0034] The optional amino acid residue for X may be the same as those mentioned above. X is preferably a valine residue.

[0035] In the formula (I), m is preferably 3 or 4, and more preferably 4.

[0036] In one embodiment, the peptide (PI) preferably consists of the amino acid sequence set forth in SEQ ID NO: 2. LL-(VPGXG) ４ (SEQ ID NO: 2) [In the formula, L represents a leucine residue, V represents a valine residue, P represents a proline residue, G represents a glycine residue, and X represents any amino acid residue other than a proline residue.]

[0037] X in the amino acid sequence of SEQ ID NO: 2 may be the same as those described above, and is preferably a valine residue.

[0038] In one embodiment, the peptide (PI) preferably consists of the amino acid sequence set forth in SEQ ID NO: 3. LL-(VPGVG) ４ (SEQ ID NO: 3) [In the formula, L represents a leucine residue, V represents a valine residue, P represents a proline residue, and G represents a glycine residue.]

[0039] An appropriate amount of peptide (P-I) is dissolved in water or a buffer solution to form an aqueous solution, and the solution is heated to a temperature equal to or higher than the transition temperature, whereby droplets are formed by liquid-liquid phase separation. When a hydrophobic substance is present in the aqueous solution, the hydrophobic substance is selectively incorporated into the droplets. The droplets can be separated and recovered by centrifugation or the like. The hydrophobic substance incorporated into the droplets can be recovered by disrupting the droplets. Therefore, peptide (P-I) can be used to concentrate hydrophobic substances. Alternatively, it can be used to separate and recover hydrophobic substances. Therefore, peptide (P-I) can be used as a concentrator for hydrophobic substances or an extractant for hydrophobic substances.

[0040] An example of a hydrophobic substance is a denatured protein. A "denatured protein" is a protein whose tertiary structure has been changed by physical or chemical action. An example of a denatured protein is a reduced denatured protein whose disulfide bonds have been reduced by a reducing agent or the like.

[0041] In one embodiment, peptide (P) may be a peptide containing at least one cysteine ​​residue (hereinafter also referred to as "peptide (P-II)"). The number of cysteine ​​residues contained in peptide (P-II) may be, for example, 1 to 10, and may be 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

[0042] The position of the cysteine ​​residue in peptide (P-II) may be, for example, the N-terminus, the C-terminus, or an internal portion of the peptide. When peptide (P-II) has amino acid sequence (1), the cysteine ​​residue may be, for example, located at either the N-terminus or the C-terminus of amino acid sequence (1), or may be located at both the N-terminus and the C-terminus of amino acid sequence (1). When peptide (P-II) has two or more copies of amino acid sequence (1), the cysteine ​​residue may be located between the multiple copies of amino acid sequence (1).

[0043] When peptide (P-II) has the amino acid sequence (1) and a hydrophobic aliphatic amino acid residue at the N-terminus and / or C-terminus, a cysteine ​​residue may be present, for example, between the amino acid sequence (1) and the hydrophobic aliphatic amino acid residue.

[0044] In one embodiment, the peptide (P-II) is preferably a peptide consisting of an amino acid sequence represented by the following general formula (II): (Z1) ｎ１ -(Cx) ｐ１ - (VPGXG) ｍ1 -(Cy) ｐ２ - (VPGXG) ｍ２ -(Cx) ｐ３ -(Z2) ｎ２ (II) [In the formula, Z1 and Z2 each independently represent a hydrophobic aliphatic amino acid residue. V represents a valine residue, P represents a proline residue, G represents a glycine residue, and X represents any amino acid residue other than a proline residue. Cx, Cy, and Cz each independently represent 1 to 5 amino acid residues including at least one cysteine ​​residue. n1 and n2 each independently represent an integer of 0 to 3, and n1 + n2 ≧ 1. m1 and m2 each independently represent an integer of 0 to 5, and 3 ≦ m1 + m2 ≦ 5. p1, p2, and p3 each independently represent 0 or 1.]

[0045] In the formula (II), Z1, Z2, n1, n2, and X are the same as Z1, Z2, n1, n2, and X in the formula (I).

[0046] In the formula (II), m1 and m2 are preferably 0 to 4, more preferably 1 to 4, still more preferably 2 or 3, and particularly preferably 2. Examples of combinations of m1 and m2 include m1=0 and m2=5, 4, or 3; m1=1 and m2=4, 3, or 2; m1=2 and m2=3, 2, or 1; m1=3 and m2=2, 1, or 0; m1=4 and m2=1 or 0; and m1=5 and m2=0. In one embodiment, m1=2 and m2=2.

[0047] In formula (II), the number of cysteine ​​residues contained in Cx, Cy, and Cz may be, for example, 1 to 5, 1 to 4, 1 to 3, or 1 or 2. The amino acid residues other than the cysteine ​​residues contained in Cx, Cy, and Cz are not particularly limited, and examples thereof include histidine residues, aspartic acid residues, glutamic acid residues, lysine residues, arginine residues, serine residues, threonine residues, aspartic acid residues, glutamine residues, tyrosine residues, glycine residues, alanine residues, proline residues, valine residues, leucine residues, isoleucine residues, methionine residues, phenylalanine residues, tryptophan residues, ornithine residues, and selenocysteine ​​residues. The amino acid residues other than the cysteine ​​residues contained in Cx, Cy, and Cz may be, for example, hydrophilic amino acid residues. Examples of hydrophilic amino acid residues include histidine residues, aspartic acid residues, glutamic acid residues, lysine residues, arginine residues, serine residues, threonine residues, asparagine residues, glutamine residues, tyrosine residues, ornithine residues, and selenocysteine ​​residues. The amino acid residues other than the cysteine ​​residues contained in Cx, Cy, and Cz may be, for example, hydrophobic amino acid residues. Examples of hydrophobic amino acid residues include glycine residues, alanine residues, valine residues, methionine residues, leucine residues, isoleucine residues, phenylalanine residues, tryptophan residues, and proline residues.

[0048] The number of amino acid residues in Cx, Cy, and Cz may be, for example, 1 to 4, 1 to 3, 1 to 2, or 1. Examples of Cx, Cy, and Cz include amino acid sequences represented by the following general formula (III): (X １ )ｑ１ C(X ２ ) ｑ２ C(X ３ ) ｑ３ (III) [wherein, X １ , X ２ , and X ３ each independently represents an amino acid residue; q1, q2, and q3 each independently represent an integer of 0 to 4.

[0049] In the formula (III), X １ , X ２ , and X ３ Examples of X include amino acid residues derived from amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, proline, and valine. Of these, X is preferably an amino acid residue selected from the group consisting of glycine residues and histidine residues.

[0050] In the formula (III), q2 is preferably an integer of 1 to 4, more preferably an integer of 1 to 3, and even more preferably 1 or 2.

[0051] In the formula (III), q1 and q3 are preferably integers of 0 to 3, more preferably integers of 0 to 2, and even more preferably 0 or 1.

[0052] Specific examples of Cx, Cy, and Cz include CGHC.

[0053] In the formula (II), p1, p2, and p3 may be, for example, all 1 (p1 = 1, p2 = 1, p3 = 1), any two may be 1 and the other one may be 1 (p1 = 1, p2 = 1, p3 = 0; p1 = 1, p2 = 0, p3 = 1; or p1 = 0, p2 = 1, p3 = 1), any one may be 1 and the other two may be 0 (p1 = 1, p2 = 0, p3 = 0; p1 = 0, p2 = 1, p3 = 0; or p1 = 0, p2 = 0, p3 = 1). 1 ≦ p1 + p2 + p3 ≦ 2 is preferred, and p1 + p2 + p3 = 1 is more preferred.

[0054] In one embodiment, the peptide (P-II) preferably consists of the amino acid sequence set forth in SEQ ID NO: 4. LL-(VPGXG) ２ -CGHC-(VPGXG) ２ (SEQ ID NO: 4) [In the formula, L represents a leucine residue, V represents a valine residue, P represents a proline residue, G represents a glycine residue, C represents a cysteine ​​residue, H represents a histidine residue, and X represents any amino acid residue other than a proline residue.]

[0055] X in the amino acid sequence of SEQ ID NO: 4 may be the same as those described above, and is preferably a valine residue.

[0056] In one embodiment, the peptide (P-II) preferably consists of the amino acid sequence set forth in SEQ ID NO: 5. LL-(VPGVG) ２ -CGHC-(VPGVG) ２ (SEQ ID NO: 5) [In the formula, L represents a leucine residue, V represents a valine residue, P represents a proline residue, G represents a glycine residue, C represents a cysteine ​​residue, and H represents a histidine residue.]

[0057] Peptide (P-II) contains a cysteine ​​residue, which can promote folding of denatured proteins, including those whose disulfide bonds have been reduced.

[0058] An appropriate amount of peptide (P-II) is dissolved in water or a buffer solution to form an aqueous solution, and the solution is heated to a temperature equal to or higher than the transition temperature, whereby droplets are formed by liquid-liquid phase separation. When a denatured protein is present in the aqueous solution, the denatured protein is selectively incorporated into the droplets. The denatured protein is folded within the droplets to become a protein in its native structure. The folded protein migrates out of the droplets. Therefore, the folded protein can be efficiently recovered by recovering the aqueous phase by centrifugation or the like. Therefore, peptide (P-II) can be used for protein folding and can be used as a protein folding agent.

[0059] Peptide (P-II) can efficiently form droplets when used in combination with peptide (PI).

[0060] Peptide (P) is not very hydrophobic, so it is easy to prepare an aqueous solution. Furthermore, because it has a low molecular weight, the viscosity of the aqueous solution is not too high, and there is no problem with molecular weight distribution. Furthermore, it can be produced by a general solid-phase peptide synthesis method. Furthermore, if it is composed only of natural amino acid residues, it can be made into a peptide with excellent biocompatibility and biodegradability.

[0061] <Droplet Formation Kit> A second aspect of the present invention is a droplet formation kit containing a peptide (P).

[0062] The peptide (P) included in the kit of this embodiment may be peptide (P-II), or may be a peptide (P) other than peptide (P-II). The peptide (P) other than peptide (P-II) is preferably peptide (P-I). When the kit of this embodiment includes peptide (P) other than peptide (P-II), the kit of this embodiment can be used as a kit for concentrating a hydrophobic substance or a kit for extracting a hydrophobic substance. When the kit of this embodiment includes peptide (P-II), the kit of this embodiment can be used as a kit for protein folding. Examples of the hydrophobic substance include denatured proteins. Examples of the denatured proteins include denatured proteins whose disulfide bonds have been reduced.

[0063] When the kit of this embodiment includes peptide (P-II), it may further include a peptide (P) other than peptide (P-II). For example, the kit of this embodiment may include peptide (P-II) and peptide (PI).

[0064] The kit of this embodiment may include other components in addition to the peptide (P), such as an aqueous solvent for dissolving the peptide (P).

[0065] Examples of aqueous solvents include water (pure water or ultrapure water such as distilled water, ion-exchanged water, or reverse osmosis water), buffer solutions, etc. The buffer solution is not particularly limited, and solutions commonly used in biochemical tests and the like can be used without particular limitation. Examples of buffer solutions include, but are not limited to, Tris-HCl buffer, phosphate buffer, phosphate-buffered saline, acetate buffer, citrate buffer, and HEPES buffer. Among these, Tris-HCl buffer is preferred as the aqueous solvent, and sodium chloride-containing Tris-HCl buffer is more preferred. A specific example of the solvent is 50 mM Tris-HCl buffer (pH 7.5) containing 0.3 M sodium chloride. The kit of this embodiment may also include a reagent for preparing the aqueous solvent. For example, when preparing a sodium chloride-containing Tris-HCl buffer as the aqueous solvent, the kit of this embodiment may also include sodium chloride, trishydroxyaminomethane, and hydrochloric acid.

[0066] The kit of this embodiment may include containers (test tubes, plastic tubes, beakers, flasks, microtubes, etc.) used for preparing the peptide solution, instructions, etc.

[0067] The kit of this embodiment can be used to prepare droplets.

[0068] <Method for forming droplets> A third aspect of the present invention is a method for forming droplets, comprising the steps of: (A) preparing an aqueous solution containing a peptide (P); and (B) incubating the aqueous solution at a temperature higher than the transition temperature of the peptide (P).

[0069] (Step (A)) In step (A), an aqueous solution containing peptide (P) is prepared.

[0070] An aqueous solution containing peptide (P) can be prepared by dissolving peptide (P) in an aqueous solvent. Peptide (P) may be peptide (P-II), or a peptide (P) other than peptide (P-II), or peptide (P-II) and a peptide (P) other than peptide (P-II) may be used in combination. As peptide (P) other than peptide (P-II), peptide (P-I) is preferred.

[0071] Examples of aqueous solvents include those listed in the "Droplet Formation Kit" section above. A preferred aqueous solvent is Tris-HCl buffer, and a sodium chloride-containing Tris-HCl buffer is more preferred. A specific example of an aqueous solvent is 50 mM Tris-HCl buffer (pH 7.5) containing 0.3 M sodium chloride.

[0072] The peptide (P) is preferably dissolved in an aqueous solvent to a concentration of 2% by mass or more. The concentration of the peptide (P) in the aqueous solution may be 2 to 15% by mass, preferably 2 to 10% by mass, more preferably 2 to 8% by mass, and even more preferably 2 to 5% by mass. When the peptide (P) is difficult to dissolve, the peptide (P) may be dissolved in the aqueous solvent while heating the aqueous solvent using a water bath or the like. The peptide (P) may also be dissolved while stirring the aqueous solvent using a stirrer, vortex, ultrasonic cleaner, or the like. When peptide (P-II) and a peptide (P) other than peptide (P-II) are used in combination, the peptide (P) other than peptide (P-II) may be adjusted to a concentration of 2% by mass or more, and the peptide (P-II) may be adjusted to a concentration of less than 2% by mass. In this case, the concentration of peptide (P-II) can be, for example, 0.001 to 1.5% by mass, 0.01 to 1.0% by mass, 0.05 to 0.5% by mass, or 0.10 to 0.5% by mass.

[0073] (Step (B)) In step (B), the aqueous solution prepared in step (A) is incubated at a temperature higher than the transition temperature of peptide (P).

[0074] For incubation, a device with a temperature maintenance function, such as an incubator, can be used. Since the transition temperature varies depending on the amino acid sequence of the peptide (P), the incubation temperature can be set according to the transition temperature of the peptide (P). The incubation temperature can be set, for example, to a temperature 1 to 10°C higher than the transition temperature of the peptide (P). The incubation time may be any time sufficient for droplets to be formed by liquid-liquid phase separation. Examples of the incubation time include 10 seconds to 5 minutes. The incubation time may be 30 seconds to 5 minutes, or 1 minute to 5 minutes.

[0075] The particle size of the droplets formed by the droplet formation method of this embodiment is not particularly limited, but may be, for example, 1 to 100 μm, 5 to 80 μm, 10 to 60 μm, or 20 to 50 μm.

[0076] In the droplet formation method of this embodiment, the use of peptide (P) prevents the viscosity of the aqueous solution from becoming too high. Furthermore, unlike polymeric materials such as polyethylene glycol, peptide (P) has no molecular weight distribution, allowing droplets to be formed with stable quality.

[0077] <Method for Concentrating Hydrophobic Substances> A fourth aspect of the present invention is a method for concentrating hydrophobic substances, which includes the steps of: (A1) preparing an aqueous solution containing a peptide (P) and a hydrophobic substance; and (B1) incubating the aqueous solution at a temperature below the phase transition temperature of the peptide (P).

[0078] (Step (A1)) In step (A1), an aqueous solution containing the peptide (P) and a hydrophobic substance is prepared.

[0079] Preferably, peptide (P) is not peptide (P-II), and more preferably peptide (PI).

[0080] The hydrophobic substance is not particularly limited. Examples of hydrophobic substances include denatured proteins, hydrophobic polymers, hydrophobic drugs, etc. Denatured proteins are proteins in which a native protein is denatured with a denaturant. Examples of denaturants include reducing agents. Examples of denatured proteins include reduced denatured proteins in which one or more disulfide bonds are reduced. The aqueous solution in step (A1) can be prepared by adding a hydrophobic substance to an aqueous solution containing peptide (P). The aqueous solution containing peptide (P) can be prepared by a method similar to step (A) of the droplet formation method described above. The hydrophobic substance may be added directly to the aqueous solution, or may be dissolved in an appropriate solvent and added to the aqueous solution. The hydrophobic substance may be added to the aqueous solution as a mixture with another substance (e.g., a hydrophilic substance). When the hydrophobic substance is a denatured protein, the denatured protein may be added to the aqueous solution as a mixture with a native protein. The concentration of the hydrophobic substance in the aqueous solution is not particularly limited. Examples of the concentration of the hydrophobic substance include 1 to 10,000 μM.

[0081] (Step (B1)) In step (B1), the aqueous solution prepared in step (A1) is incubated at a temperature higher than the transition temperature of peptide (P). Step (B1) can be performed in the same manner as step (B) in the droplet formation method described above. The hydrophobic substance is selectively incorporated into the droplets formed in step (B1), allowing the hydrophobic substance to be concentrated in the droplets.

[0082] (Other Steps) The method for concentrating a hydrophobic substance of this embodiment may include other steps in addition to the steps (A1) and (B1) described above. Examples of the other steps include (C1) recovering droplets from the aqueous solution after the step (B1) and (D1) recovering the hydrophobic substance from the droplets.

[0083] <Step (C1)> After step (B1), droplets may be recovered from the aqueous solution. The droplets can be recovered by centrifugation or the like. The rotation speed of the centrifugation may be, for example, 5,000 to 10,000 rpm. The centrifugation time may be, for example, 10 seconds or more, and may be 20 seconds or more. From the viewpoint of preventing the droplets from being destroyed, the centrifugation time is preferably 5 minutes or less, more preferably 3 minutes or less, and even more preferably 1 minute or less. The centrifugation time may be 10 seconds or more and 5 minutes or less, 20 seconds or more and 3 minutes or less, or 20 seconds or more and 1 minute or less. The droplets can be recovered as a precipitate from the centrifugation.

[0084] <<Step (D1)>> The droplets recovered in step (C1) may be disrupted to recover the hydrophobic substance. Since the hydrophobic substance is incorporated into the droplets without agglomerating, the recovered hydrophobic substance can be used for various purposes.

[0085] The hydrophobic substance concentration method of this embodiment allows hydrophobic substances to be concentrated in droplets while suppressing aggregation. Many protein pharmaceuticals, such as antibodies and insulin, form quaternary structures in which multiple polypeptide chains are complexed. Promoting this formation is important for improving the synthesis efficiency of protein pharmaceuticals and reducing production costs. To form a quaternary structure, it is necessary to concentrate multiple polypeptide chains while suppressing aggregation. The hydrophobic substance concentration method of this embodiment allows hydrophobic substances, such as reduced and denatured proteins, to be concentrated in droplets while suppressing aggregation. Therefore, droplets may be used as a site where multiple polypeptides form quaternary structures. This method is also expected to contribute to the development of materials that promote quaternary structure formation.

[0086] In the method for concentrating a hydrophobic substance of this embodiment, the hydrophobic substance is concentrated in droplets and the droplets are recovered, thereby separating and recovering the hydrophobic substance.

[0087] <Protein Folding Method> A fifth aspect of the present invention is a protein folding method, which includes the steps of: (A2) preparing an aqueous solution containing a peptide (P) and a protein; and (B2) incubating the aqueous solution at a temperature higher than the transition temperature of the peptide.

[0088] (Step (A2)) In step (A2), an aqueous solution containing the peptide (P) and a protein is prepared.

[0089] The peptide (P) is preferably peptide (P-II). The peptide (P) may be a combination of peptide (P-II) and a peptide (P) other than peptide (P-II). The peptide (P) other than peptide (P-II) is preferably peptide (PI).

[0090] The protein is a protein to be folded, and is preferably a denatured protein, preferably a reduced denatured protein in which one or more disulfide bonds have been reduced.

[0091] The protein may be an unfolded protein or a misfolded protein. An unfolded protein is a protein that is not folded. In an unfolded protein, disulfide bonds formed in a protein in its native structure 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 and the protein has a structure other than the native structure. A protein in its native structure is usually the thermodynamically most stable structure. However, a misfolded protein is folded into a structure other than the native structure. The protein may be a protein having at least one type selected from the group consisting of a disulfide bond and a reduced disulfide bond.

[0092] The aqueous solution in step (A2) can be prepared by adding a protein to an aqueous solution containing peptide (P). The aqueous solution containing peptide (P) can be prepared by the same method as step (A) of the above-mentioned <Droplet Formation Method>. The aqueous solution containing peptide (P) can be prepared by dissolving peptide (P) in an aqueous solvent. Examples of aqueous solvents include those listed in the above-mentioned <Droplet Formation Kit> section. As the aqueous solvent, Tris-HCl buffer is preferred, and sodium chloride-containing Tris-HCl buffer is more preferred. A specific example of the aqueous solvent is 50 mM Tris-HCl buffer (pH 7.5) containing 0.3 M sodium chloride.

[0093] The concentration of peptide (P) in the aqueous solution containing peptide (P) may be any concentration capable of promoting protein folding, and may be, for example, 0.001% by mass or more. Examples of the concentration of peptide (P) include 0.005% by mass or more, 0.01% by mass or more, 0.05% by mass or more, and 0.10% by mass or more. When peptide (P) is peptide (P-II) alone, the concentration of peptide (P) in the aqueous solution may be, for example, 2% by mass or less, 1.5% by mass or less, 1.0% by mass or less, or 0.5% by mass or less. In this case, the concentration of peptide (P-II) in the aqueous solution may be, for example, 0.001 to 1.5% by mass, 0.01 to 1.0% by mass, 0.05 to 0.5% by mass, or 0.10 to 0.5% by mass. When peptide (P-II) and a peptide (P) other than peptide (P-II) are used in combination as peptide (P), the concentration of the peptide (P) other than peptide (P-II) may be, for example, 2% by mass or more. The concentration of the peptide (P) other than peptide (P-II) in the aqueous solution may be, for example, 2 to 15% by mass, preferably 2 to 10% by mass, more preferably 2 to 8% by mass, and even more preferably 2 to 5% by mass. In this case, the concentration of peptide (P-II) may be the same as that described above.

[0094] The protein may be added to the aqueous solution as is, or may be dissolved in an appropriate solvent and then added to the aqueous solution. The protein concentration in the aqueous solution is not particularly limited. Examples of the protein concentration include 1 to 10,000 μM.

[0095] (Step (B2)) In step (B2), the aqueous solution prepared in step (A2) is incubated. The incubation temperature may be any temperature that allows protein folding. Examples of the incubation temperature include 10 to 70°C, preferably 20 to 65°C, and more preferably 30 to 65°C.

[0096] In step (B2), when droplets are formed in the aqueous solution prepared in step (A2), the incubation temperature can be set to a temperature higher than the transition temperature of the peptide (P). Since the transition temperature varies depending on the amino acid sequence of the peptide (P), the incubation temperature can be set according to the transition temperature of the peptide (P). The incubation temperature can be set, for example, to a temperature 1 to 10°C higher than the transition temperature of the peptide (P). When droplets are formed, unfolded proteins are incorporated into the droplets. Meanwhile, folded proteins migrate out of the droplets. This allows the folded protein to be separated from the unfolded protein by separating the droplets from the aqueous phase.

[0097] When peptide (P-II) and a peptide (P) other than peptide (P-II) are used in combination as peptide (P), the incubation temperature may be set to a temperature equal to or higher than the transition temperature of the peptide (P) other than peptide (P-II). By setting the temperature equal to or higher than the transition temperature of the peptide (P) other than peptide (P-II), liquid-liquid phase separation is promoted by the peptide (P) other than peptide (P-II), and droplets are formed.

[0098] The incubation time may be any time sufficient for the protein to be folded. Examples of the incubation time include 5 minutes or more, 10 minutes or more, 15 minutes or more, 30 minutes or more, 45 minutes or more, or 60 minutes or more. The incubation time can be, for example, 5 to 180 minutes, 10 to 180 minutes, 15 to 180 minutes, 30 to 180 minutes, 45 to 180 minutes, or 60 to 180 minutes.

[0099] (Other Steps) The protein folding method of this embodiment may include other steps in addition to the above steps (A2) and (B2). Examples of such other steps include (C2) recovering the folded protein from the aqueous solution after step (B2).

[0100] <<Step (C2)>> After step (B2), the folded protein may be recovered from the aqueous solution. Most of the protein is converted into a folded protein by step (B2). Therefore, the folded protein can be recovered by applying a known purification method such as reverse-phase high-performance liquid chromatography to the aqueous solution after step (B2).

[0101] When droplets are formed in step (B2), the folded protein migrates to the aqueous phase. Therefore, the folded protein can be recovered from the aqueous phase. Separation of the aqueous phase and the droplets can be performed by centrifugation or the like. The rotation speed of the centrifugation can be, for example, 5,000 to 10,000 rpm. The centrifugation time can be, for example, 10 seconds or more, and may be 20 seconds or more. From the viewpoint of preventing the droplets from being destroyed, the centrifugation time is preferably 5 minutes or less, more preferably 3 minutes or less, and even more preferably 1 minute or less. The centrifugation time may be 10 seconds or more and 5 minutes or less, 20 seconds or more and 3 minutes or less, or 20 seconds or more and 1 minute or less. The aqueous phase can be recovered as the supernatant of the centrifugation. After recovering the aqueous phase as the supernatant, the folded protein may be further purified using a known purification method such as reverse-phase high-performance liquid chromatography.

[0102] The protein folding method of this embodiment can promote protein folding involving disulfide bond formation with extremely high efficiency (e.g., 90% or more). Protein pharmaceuticals such as insulin and antibodies contain disulfide bonds. The protein folding method of this embodiment can be applied to the folding of such proteins. Therefore, it is expected to be applied to the production of protein pharmaceuticals.

[0103] Peptides (P), especially peptide (P-II), have the potential to serve as a basis for materials that promote protein quaternary structure formation. Many protein pharmaceuticals, such as antibodies and insulin, form quaternary structures composed of multiple polypeptide chains. Promoting this structure is important for increasing the synthesis efficiency of protein pharmaceuticals and reducing production costs. However, no artificial materials have been shown to promote protein quaternary structure formation. Promoting protein quaternary structure formation requires concentrating multiple polypeptide chains while suppressing aggregation. The aqueous two-phase system formed by peptide (P) has the effect of capturing and concentrating denatured proteins while suppressing aggregation. Furthermore, the use of peptide (P-II) promotes protein folding within droplets. Therefore, protein folding methods using peptide (P) are expected to improve the efficiency of protein pharmaceutical production, such as antibodies and insulin.

[0104] Peptides (P) composed only of natural amino acid residues are expected to have excellent biocompatibility. Accumulation of denatured proteins in vivo leads to amyloid formation, which is the cause of various diseases such as neurodegenerative diseases, type 2 diabetes, and amyotrophic lateral sclerosis (ALS). Peptides (P) are also expected to be applicable as pharmaceuticals that suppress the accumulation of denatured proteins in vivo.

[0105] The present invention will be described below with reference to experimental examples, but the present invention is not limited to the following experimental examples. In the following experimental examples, mass % may be expressed as "wt %".

[0106] Experimental Example 1 Preparation of Modified BPTI Reagents Bovine pancreatic trypsin inhibitor (BPTI) was purchased from Pro-Spec-Tany TechnoGene (Rehovot, Israel). 1,4-Dithiothreitol and urea were purchased from Nacalai Tesque, Inc. (Kyoto, Japan).

[0107] (Experimental Procedure) BPTI (10 mg) dissolved in 0.1 M Tris-HCl (pH 8.0, 1.0 mL) containing 30 mM 1,4-dititothreitol and 8 M urea was incubated at 50°C for 3 hours. Completely reduced and denatured BPTI was purified by HPLC using a PU-4180 pump and UV-4075 detector manufactured by JASCO Corporation (Tokyo, Japan) on an InertSustain C18 column (4.6 mmφ×250 mm) manufactured by GL Sciences (Tokyo, Japan) at a flow rate of 1.0 mL min. －１ The denatured BPTI fraction was collected and freeze-dried. The resulting powder was stored at -30°C until use.

[0108] <Synthesis of LL-ELP4> (Reagents) Acetic anhydride was purchased from Kanto Chemical Co., Ltd. (Tokyo, Japan). Acetonitrile, N,N'-dimethylformamide (DMF), diethyl ether (Et ２N,N-diisopropylethylamine (DIEA) was purchased from Nacalai Tesque (Kyoto, Japan). Triisopropylsilane (TIS) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Fmoc-NH-SAL Resin, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Val-OH, Fmoc-Leu-OH, 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU), and 1,2,3-benzotriazol-1-ol monohydrate (HOBt・H ２ 0) was purchased from Watanabe Chemical Industry Co., Ltd. (Hiroshima, Japan). Ultrapure water for HPLC (filtered through a 0.22 μm membrane filter, >18.2 MΩcm) was purified using a Purelab DV35 manufactured by ELGA (Buckinghamshire, UK).

[0109] (Experimental Procedure) LL-ELP4 (LL-(VPGVG) ４ ) was synthesized using a fully automated microwave peptide synthesizer (Initiator+Alstra) from Biotage (Uppsala, Sweden) under the following conditions: Coupling: 75°C for 5 minutes; Fmoc group deprotection: first reaction at 25°C for 3 minutes, second reaction at 25°C for 10 minutes; N-terminal acetylation: 25°C for 10 minutes; Washing: 4 times with DMF after coupling, and 4 times with DMF after the second deprotection.

[0110] For the coupling, deprotection, and acetylation reactions, 0.5 M DMF solutions of each amino acid, HOBt, and HBTU, 2 M NMP solution of DIEA, 20% piperidine in DMF, and 5 M DMF solution of acetic anhydride were prepared. To cleave the peptide from the resin, a deprotection cocktail containing TIS (62.5 μL), TFA (2.375 mL), and water (62.5 μL) was used. The deprotection cocktail (2.5 mL) was added to the resin, and the reaction tube was left at 25°C for 90 minutes, with gentle shaking every 30 minutes. The mixture was filtered, and the solution was collected in a polypropylene centrifuge. The reaction tube was washed with TFA (500 μL, three times), which was also collected by filtration. The centrifuge tube was filled with Et ２ 0 (40 mL) was added, the mixture was vortexed for 1 minute, and centrifuged (3500 × g, 4°C, 5 minutes) using a micro-refrigerated centrifuge (Model 3700) manufactured by Kubota Shoji Co., Ltd. (Tokyo, Japan). The supernatant was then removed. This procedure was repeated three times, and the peptide was vacuum-dried for 2 hours at 25°C, dispersed in water, and lyophilized using a Tokyo Rikaki Co., Ltd. (Tokyo, Japan) freeze-drier (FDU-1200) equipped with an oil-sealed rotary vacuum pump (GLD-051) manufactured by ULVAC, Inc. (Tokyo, Japan). The synthesized peptides were purified using a PU-4086-Binary pump, a UV-4075 detector, and a CHF122SC fraction collector manufactured by JASCO Corporation (Tokyo, Japan), a TA12S05-2520WX Actus Triart column (20 mmφ×250 mm) manufactured by YMC Co., Ltd. (Tokyo, Japan), and a flow rate of 18.9 mL min －１ The product was purified by semi-preparative reversed-phase high performance liquid chromatography (RP-HPLC) (see Figure 1). The gradient profile of the semi-preparative RP-HPLC was water / acetonitrile = 70 / 30 (0 min), 50 / 50 (20 min), with a linear gradient from 0 to 20 min.

[0111] The collected samples were freeze-dried and stored at -20°C. The structures of the synthesized peptides were determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). Instrument information: Bruker (Bremen, Germany) autoflex speed spectrometer, 2,5-dihydroxybenzoic acid was used as the matrix, and the measurement was performed in reflector positive mode.

[0112] LL-ELP4 Na ＋ A signal at m / z 1945.147 corresponding to the complex of Na ＋ Monoisotopic mass: 1945.125 (C ９０ H １５１ N ２３ O ２３ )) (see Figure 2).

[0113] The structures of LL-ELP4 and FF-ELP4, which was synthesized as a control compound, are shown below.

[0114]

[0115] <Transmittance Measurement> (Experimental Procedure) FF-ELP4 or LL-ELP4 was dissolved in 50 mM Tris-HCl (pH 7.5) containing 0.3 M sodium chloride and incubated at 60°C. LL-ELP4 was dissolved in 50 mM Tris-HCl (pH 7.5) to a final concentration of 2 wt%. FF-ELP4 could not be dissolved to a final concentration of 2 wt%, so it was dissolved in 50 mM Tris-HCl (pH 7.5) to a final concentration of 1 wt%.

[0116] The transmittance was measured using a UV-visible spectrophotometer V-650 manufactured by JASCO Corporation (Tokyo, Japan). The temperature of the sample prepared as described above was changed, and the absorbance at a wavelength of 600 nm was monitored. A quartz cell (optical path length: 10 mm) manufactured by Sansho Co., Ltd. was used as the measurement cell.

[0117] As an actual observation, the sample prepared as described above was observed for changes in turbidity when the temperature was changed, and photographs were taken.

[0118] (Results) The results are shown in Figure 3. In Figure 3, a graph showing the results of monitoring transmittance is shown together with photographs of actual observations. The transition temperature of a 1 wt% solution of FF-ELP was 44°C. The transition temperature of a 2 wt% solution of LL-ELP was 53°C.

[0119] <Phase-contrast microscope observation> (Experimental procedure) Phase-contrast microscope observation was performed using an IX-73 microscope manufactured by Olympus Corporation (Tokyo, Japan). A sample was dropped onto a glass slide, and a cover glass was placed over the sample via a 0.2 mm thick silicone spacer. The sample was observed while incubating at 60°C. Samples were prepared by dissolving FF-ELP4 or LL-ELP4 in 50 mM Tris-HCl (pH 7.5) containing 0.3 M sodium chloride. FF-ELP4 was dissolved to a final concentration of 1 wt%. LL-ELP4 was dissolved to a final concentration of 5 wt%.

[0120] (Results) Phase-contrast microscopic images are shown in Figure 4. In the 5 wt% solution of LL-ELP4, droplets of moderate size were observed. On the other hand, in the 1 wt% solution of FF-ELP4, only very small droplets were observed.

[0121] <Evaluation of uptake of denatured BPTI> (Experimental procedure) FF-ELP4 or LL-ELP4 was dissolved in 50 mM Tris-HCl (pH 7.5) containing 0.3 M sodium chloride and preincubated for 1 minute at 60° C. Fully reduced denatured BPTI (170 μM) or native BPTI (170 μM) was dissolved in 1.6 M urea containing 0.05% TFA to prepare a fully reduced denatured BPTI solution and a native BPTI solution. To the preincubated solution, a fully reduced denatured BPTI solution or a native BPTI solution was added (final concentrations: fully reduced denatured BPTI or native BPTI: 30 μM; urea: 70 mM; FF-ELP4: 1 wt%; LL-ELP4: 5 wt%; Tris-HCl: 50 mM; NaCl: 0.3 M, pH 7.5). This was incubated overnight at 60°C and then centrifuged (8,000 rpm, 25°C, 30 seconds) in a Taiyo Co., Ltd. (Osaka, Japan) high-spin mini centrifuge HSC-12000 to quickly separate the supernatant and precipitate (droplets). The reaction was stopped by adding 400 μL of 1 M HCl aqueous solution to each of the supernatant and precipitate. The supernatant and the precipitate were subjected to HPLC analysis to quantify the amount of reduced modified BPTI or native BPTI. The analysis was performed using a Primeide HPLC system (1410 UV detector, 1310 column oven, 1110 pump) manufactured by Hitachi, Ltd. (Tokyo, Japan) and a TSKgel Protein C4-300 column (4.6 mmφ×150 mm) manufactured by Tosoh Corporation (Tokyo, Japan) at a flow rate of 1.0 mL min －１ The analysis was carried out by reversed-phase high-performance liquid chromatography (RP-HPLC) (solvent A: 0.05% TFA water, solvent B: 0.05% TFA acetonitrile; proportion of solvent A: 95% at 0 min, 80% at 15 min, 30% at 115 min, linear gradient).

[0122] (Results) The results are shown in Table 1.

[0123]

[0124] In a 5 wt% LL-ELP4 solution, reduced denatured BPTI was confirmed to be concentrated at a high concentration within the droplets. The reduced denatured BPTI recovered from the droplets could be redispersed in water, confirming that aggregation was suppressed. On the other hand, most of the native BPTI was present outside the droplets. In a 1 wt% FF-ELP4 solution, reduced denatured BPTI tended to be concentrated within the droplets. On the other hand, most of the native BPTI was present outside the droplets. These results demonstrate that the droplets formed in the LL-ELP4 solution are capable of concentrating reduced denatured proteins to a high concentration.

[0125] [Experimental Example 2] <Preparation of Modified BPTI> Modified BPTI was prepared in the same manner as in Experimental Example 1.

[0126] <Synthesis of LL-ELP4-SS> (Reagents) Acetic anhydride was purchased from Kanto Chemical Co., Ltd. (Tokyo, Japan). Hydrogen peroxide, acetonitrile, N,N'-dimethylformamide (DMF), diethyl ether (Et ２ N,N-methyl-2-pyrrolidone (NMP), piperidine, and trifluoroacetic acid (TFA) were purchased from Kishida Chemical Co., Ltd. (Tokyo, Japan). N,N-diisopropylethylamine (DIEA) was purchased from Nacalai Tesque Co., Ltd. (Kyoto, Japan). Triisopropylsilane (TIS) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Fmoc-NH-SAL Resin, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Val-OH, Fmoc-Leu-OH, 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU), and 1,2,3-benzotriazol-1-ol monohydrate (HOBt・H ２0) was purchased from Watanabe Chemical Industry Co., Ltd. (Hiroshima, Japan). Ultrapure water for HPLC (filtered through a 0.22 μm membrane filter, >18.2 MΩcm) was purified using a Purelab DV35 manufactured by ELGA (Buckinghamshire, UK).

[0127] (Experimental Procedure) LL-ELP4-SS (LL-(VPGVG) ２ -CGHC-(VPGVG) ２ ) was synthesized using a Biotage (Uppsala, Sweden) fully automated microwave peptide synthesizer (Initiator+ Alstra) under the following conditions: Coupling: 75°C for 5 minutes (except for the coupling of cysteine ​​and histidine residues, which was carried out at 25°C for 60 minutes); Deprotection of Fmoc group: First reaction at 25°C for 3 minutes, second reaction at 25°C for 10 minutes; Acetylation of N-terminus: 25°C for 10 minutes; Washing: 4 times with DMF after coupling, and 4 times with DMF after the second deprotection.

[0128] For the coupling, deprotection, and acetylation reactions, 0.5 M DMF solutions of each amino acid, HOBt, and HBTU, 2 M NMP solution of DIEA, 20% piperidine in DMF, and 5 M DMF solution of acetic anhydride were prepared. To cleave the peptide from the resin, a deprotection cocktail containing TIS (62.5 μL), TFA (2.375 mL), and water (62.5 μL) was used. The deprotection cocktail (2.5 mL) was added to the resin, and the reaction tube was left at 25°C for 90 minutes, gently shaking every 30 minutes. The mixture was filtered, and the solution was collected in a polypropylene centrifuge. The reaction tube was washed with TFA (500 μL, three times), which was also filtered and collected. Et ２0 (40 mL) was added, the mixture was vortexed for 1 minute, and centrifuged (3500 × g, 4°C, 5 minutes) using a micro-refrigerated centrifuge (Model 3700) manufactured by Kubota Shoji Co., Ltd. (Tokyo, Japan). The supernatant was then removed. This procedure was repeated three times, and the peptide was vacuum-dried for 2 hours at 25°C, dispersed in water, and lyophilized using a Tokyo Rikaki Co., Ltd. (Tokyo, Japan) freeze-drier (FDU-1200) equipped with an oil-sealed rotary vacuum pump (GLD-051) manufactured by ULVAC, Inc. (Tokyo, Japan). The synthesized peptides were purified using a PU-4086-Binary pump, a UV-4075 detector, and a CHF122SC fraction collector manufactured by JASCO Corporation (Tokyo, Japan), a TA12S05-2520WX Actus Triart column (20 mmφ×250 mm) manufactured by YMC Co., Ltd. (Tokyo, Japan), and a flow rate of 18.9 mL min －１ The semi-preparative RP-HPLC gradient profile was: water / acetonitrile = 70 / 30 (0 min), 50 / 50 (20 min), linear gradient from 0 to 20 min.

[0129] The collected samples were freeze-dried. To oxidize the cysteines in the obtained samples, the synthesized peptide was added to water at a concentration of 1 mM and hydrogen peroxide at a concentration of 5 mM, and the mixture was stirred at room temperature under atmospheric pressure for 24 hours. The reaction mixture was purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) using the same method as after peptide synthesis (Figure 6). The gradient profile of the semi-preparative RP-HPLC was: water / acetonitrile = 70 / 30 (0 min), 50 / 50 (20 min), linear gradient from 0 to 20 min.

[0130] The collected samples were freeze-dried and stored at -20°C. The structures of the synthesized peptides were determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). Instrument information: Bruker (Bremen, Germany) autoflex speed spectrometer, 2,5-dihydroxybenzoic acid used as the matrix, reflector positive mode measurement.

[0131] LL-ELP4-SS H ＋ complex and Na ＋ Signals at m / z 2321.302 and 2343.267 corresponding to the complex were observed (H ＋ , Na ＋ Monoisotopic masses: 2321.226, 2343.208 (C １０４ H １６９ N ２９ O ２７ S ２ )) (Figure 7).

[0132] The structure of LL-ELP4-SS is shown below.

[0133]

[0134] <Transmittance Measurement> (Experimental Procedure) LL-ELP4-SS was dissolved in 50 mM Tris-HCl (pH 7.5) containing 0.3 M sodium chloride and incubated at 60° C. LL-ELP4-SS was dissolved in 50 mM Tris-HCl (pH 7.5) to a final concentration of 2 wt %.

[0135] The transmittance was measured using a UV-visible spectrophotometer V-650 manufactured by JASCO Corporation (Tokyo, Japan). The temperature of the sample prepared as described above was changed, and the absorbance at a wavelength of 600 nm was monitored. A quartz cell (optical path length: 10 mm) manufactured by Sansho Co., Ltd. was used as the measurement cell.

[0136] As an actual observation, the sample prepared as described above was observed for changes in turbidity when the temperature was changed.

[0137] (Results) As a result of monitoring the transmittance, the transition temperature of a 2 wt % solution of LL-ELP4-SS was 48°C.

[0138] <Phase-contrast Microscope Observation: LL-ELP4 / LL-ELP4-SS Mixed Solution> (Experimental Procedure) Phase-contrast microscopic observation was performed using an IX-73 microscope manufactured by Olympus Corporation (Tokyo, Japan). A cover glass was placed on a slide glass, with a 0.2 mm thick silicone spacer interposed between them, and the resulting sample was incubated at 60°C for observation. The peptide solution was prepared by dissolving LL-ELP4 at 2 wt% and LL-ELP4-SS at 0.7 mM (0.16 wt%) in 50 mM Tris-HCl (pH 7.5) containing 0.3 M sodium chloride.

[0139] (Results) Phase contrast microscopic images are shown in Figure 8. Droplets of appropriate size were observed in the peptide solution.

[0140] <Folding Test of Denatured BPTI: LL-ELP4 / LL-ELP4-SS Mixed Solution> (Experimental Procedure) LL-ELP4 and LL-ELP4-SS were dissolved in a buffer (50 mM Tris-HCl, 0.3 M NaCl, pH 7.5) and preincubated at 60°C for 3 minutes. To the preincubated solution, fully reduced denatured BPTI (600 μM) dissolved in 1.6 M urea containing 0.05% TFA was added (final concentrations: BPTI: 30 μM, urea: 80 mM, LL-ELP4: 2 wt%, LL-ELP4-SS: 0.7 mM (0.16 wt%), Tris-HCl: 50 mM, NaCl: 0.3 M, pH 7.5). After incubation at 60°C for 1, 10, and 60 minutes, the mixture was centrifuged (10,000 rpm, 25°C, 10 seconds) using a high-spin mini centrifuge HSC-12000 (manufactured by Taiyo Co., Ltd., Osaka, Japan) to quickly separate the supernatant and precipitate. The reaction was stopped by adding 1 M HCl aq. to each of the supernatant and precipitate. The mixture was then purified using a Primeide HPLC system (manufactured by Hitachi, Ltd., Tokyo, Japan) (1410 UV detector, 1310 column oven, 1110 pump) with a TSKgel Protein C4-300 column (4.6 mmφ × 150 mm) (manufactured by Tosoh Corporation, Tokyo, Japan) at a flow rate of 1.0 mL min. －１ The mixture was analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) (solvent A: 0.05% TFA water, solvent B: 0.05% TFA acetonitrile; proportion of solvent A: 95% at 0 minutes, 80% at 15 minutes, 30% at 115 minutes, linear gradient).

[0141] (Results) The results are shown in Figure 9. The upper panel of Figure 9 shows the results of chromatography of the aqueous phase (outside the droplet) of the peptide solution. "N" indicates native structure BPTI. The lower panel of Figure 9 shows the results of chromatography of the reduced-denatured BPTI solution used as a reference. "R" indicates reduced-denatured BPTI. In the aqueous phase of the peptide solution, 99% or more of the added reduced-denatured BPTI was detected as native structure BPTI. These results confirmed that in the mixed peptide solution of LL-ELP4 and LL-ELP4-SS, the reduced-denatured protein could be folded into the native structure protein. Furthermore, it was confirmed that the native structure protein could be recovered in the aqueous phase.

[0142] <Phase-contrast microscope observation: LL-ELP4-SS solution> (Experimental procedure) Phase-contrast microscope observation was performed using an IX-73 microscope manufactured by Olympus Corporation (Tokyo, Japan). A cover glass was placed on a slide glass with a 0.2 mm thick silicone spacer interposed between them, and the sample was then incubated at 60°C for observation. The peptide solution was prepared by dissolving LL-ELP4-SS at 2 wt % in 50 mM Tris-HCl (pH 7.5) containing 0.3 M sodium chloride.

[0143] (Results) A phase contrast microscope image is shown in Figure 10. Droplets of appropriate size were observed in the peptide solution.

[0144] <Folding Test of Denatured BPTI: LL-ELP4-SS Solution> (Experimental Procedure) A folding test of denatured BPTI was performed in the same manner as in the above <Folding Test of Denatured BPTI: LL-ELP4 / LL-ELP4-SS Mixed Solution>, except that LL-ELP4-SS was used alone instead of the combination of LL-ELP4 and LL-ELP4-SS. The final concentration of LL-ELP4-SS in the test solution was 0.16 wt%. Since droplets were not formed in the LL-ELP4-SS solution, centrifugation was not performed.

[0145] The results are shown in Figure 11. The upper panel of Figure 11 shows the results of chromatography of the peptide solution. "N" indicates native structure BPTI. The lower panel of Figure 11 shows the results of chromatography of the reduced-denatured BPTI solution used as a reference. "R" indicates reduced-denatured BPTI. In the peptide solution, 99% or more of the added reduced-denatured BPTI was detected as native structure BPTI. These results confirmed that the reduced-denatured protein can be folded into the native structure protein even in a peptide solution containing only LL-ELP4-SS.

[0146] These results confirmed that the folding of reduced, denatured proteins was promoted in peptide solutions containing LL-ELP4-SS. Furthermore, it was confirmed that the formation of droplets using a combination of LL-ELP4-SS and LL-ELP4 enabled the recovery of highly pure, folded, native proteins in the aqueous phase.

[0147] According to the present invention, there are provided peptides that can be used for protein concentration and recovery or protein folding, droplet formation kits containing the peptides, and droplet formation methods, hydrophobic substance concentration methods, and protein folding methods using the peptides.

[0148] Although the preferred embodiments of the present invention have been described above, the present invention is not limited to these embodiments. Addition, omission, substitution, and other modifications of the configuration are possible within the scope of the spirit of the present invention. The present invention is not limited by the above description, but is limited only by the scope of the appended claims.

Claims

1. A peptide having the following characteristics (a) to (c): (a) 2 g or more is soluble in 100 mL of 50 mM Tris-HCl buffer (pH 7.5) containing 0.3 M sodium chloride at 20° C.; (b) reversibly undergoes phase transition in the Tris-HCl buffer, and has a transition temperature of 40 to 70° C. when 2 g is dissolved in 100 mL of the Tris-HCl buffer; and (c) forms droplets at a temperature equal to or higher than the transition temperature when 2 g is dissolved in 100 mL of the Tris-HCl buffer.

2. The peptide according to claim 1, comprising the amino acid sequence set forth in SEQ ID NO:

1. VPGXG (SEQ ID NO: 1) [wherein V represents a valine residue, P represents a proline residue, G represents a glycine residue, and X represents any amino acid residue other than a proline residue.] 3. The peptide according to claim 2, which contains, in addition to the amino acid sequence set forth in SEQ ID NO:1, one or more hydrophobic aliphatic amino acid residues at at least one of the N-terminus and C-terminus.

4. The peptide according to claim 3, wherein the hydrophobic aliphatic amino acid residue is at least one selected from the group consisting of leucine residues, isoleucine residues, valine residues, alanine residues, and methionine residues.

5. The peptide according to claim 3, comprising an amino acid sequence represented by the following general formula (I): (Z1) ｎ１ - (VPGXG) ｍ - (Z2) ｎ２ (I) [In the formula, Z1 and Z2 each independently represent a hydrophobic aliphatic amino acid residue. V represents a valine residue, P represents a proline residue, G represents a glycine residue, and X represents any amino acid residue other than a proline residue. n1 and n2 each independently represent an integer of 0 to 3, and n1+n2≧1. m represents an integer of 3 to 5.] 6. The peptide according to claim 5, wherein the amino acid sequence represented by the general formula (I) is the amino acid sequence set forth in SEQ ID NO:

2. LL-(VPGXG) ４ (SEQ ID NO: 2) [In the formula, L represents a leucine residue, V represents a valine residue, P represents a proline residue, G represents a glycine residue, and X represents any amino acid residue other than a proline residue.] 7. A peptide according to any one of claims 1 to 6, which is used to enrich hydrophobic substances.

8. The peptide of claim 7, wherein the hydrophobic substance is a denatured protein.

9. The peptide of claim 1, comprising at least one cysteine ​​residue.

10. The peptide according to claim 9, comprising an amino acid sequence represented by the following general formula (II): (Z1) ｎ１ - (Cx) ｐ１ - (VPGXG) ｍ1 - (Cy) ｐ２ - (VPGXG) ｍ２ - (Cx) ｐ３ - (Z2) ｎ２ (II) [In the formula, Z1 and Z2 each independently represent a hydrophobic aliphatic amino acid residue. V represents a valine residue, P represents a proline residue, G represents a glycine residue, and X represents any amino acid residue other than a proline residue. Cx, Cy, and Cz each independently represent 1 to 5 amino acid residues including at least one cysteine ​​residue. n1 and n2 each independently represent an integer of 0 to 3, and n1+n2≧1. m1 and m2 each independently represent an integer of 0 to 5, and 3≦m1+m2≦5. p1, p2, and p3 each independently represent 0 or 1, and p1+p2+p3≧1.] 11. The peptide according to claim 10, wherein the amino acid sequence represented by the general formula (II) is the amino acid sequence set forth in SEQ ID NO:

4. LL-(VPGXG) ２ -CGHC-(VPGXG) ２ (SEQ ID NO: 4) [In the formula, L represents a leucine residue, V represents a valine residue, P represents a proline residue, G represents a glycine residue, C represents a cysteine ​​residue, H represents a histidine residue, and X represents any amino acid residue other than a proline residue.] 12. A peptide according to any one of claims 9 to 11, for use in protein folding.

13. A kit for forming droplets, comprising the peptide according to any one of claims 1 to 6 and 9 to 11.

14. A method for forming droplets, comprising: (A) preparing an aqueous solution containing a peptide according to any one of claims 1 to 6 and 9 to 11; and (B) incubating the aqueous solution at a temperature higher than the transition temperature of the peptide.

15. A method for concentrating a hydrophobic substance, comprising: (A1) preparing an aqueous solution containing a peptide according to any one of claims 1 to 6 and a hydrophobic substance; and (B1) incubating the aqueous solution at a temperature higher than the transition temperature of the peptide.

16. The method for concentrating a hydrophobic substance according to claim 15, wherein the hydrophobic substance is a denatured protein.

17. A method for folding a protein, comprising: (A2) preparing an aqueous solution containing the peptide according to any one of claims 9 to 11 and a protein; and (B2) incubating the aqueous solution.

18. The protein folding method according to claim 17, wherein the protein is a denatured protein.

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