JP6857857B2 - Peptides and their self-assembly methods, their aggregates, cell culture substrates using them, and methods for producing cell sheets. - Google Patents

Description

The present invention relates to peptides and self-assembly methods thereof, aggregates thereof, cell culture substrates using these, and methods for producing cell sheets.

Elastin is known as a protein that constitutes animal tissues including the human body. This elastin is a fibrous protein that functions extracellularly like collagen, and has the property of expanding and contracting (elasticity) like rubber, so it is involved in imparting flexibility to tissues. .. Therefore, it is widely distributed in tissues and organs that require elasticity (for example, in humans, the dermis, ligaments, tendons, lungs, blood vessel walls, etc. of the skin).

This elastin is first biosynthesized in vascular smooth muscle cells and fibroblasts as a precursor protein tropoelastin (molecular weight of about 70,000) in vivo. Tropoelastin then self-assembles around and in the interstices of glycoproteins called microfibrils and then is properly cross-linked between molecules to become insoluble elastin. This first step, the regular self-assembly of tropoelastin, is important for the formation of normal elastin in vivo, and the phenomenon of this self-assembly is called "core selvation." In other words, core selvation is deeply involved in the formation of normal elastin.

The core selvation properties of elastin can be observed in vitro (Fig. 1). That is, an aqueous solution of a series of peptides having a sequence derived from tropoelastin or elastin (elastin-derived peptide) is a transparent and uniform solution at a low temperature (25 ° C. or lower). However, when the temperature is raised above body temperature (37 ° C.), the molecules self-assemble, resulting in a cloudy solution. When the cloudy solution is cooled again, it returns to a transparent solution, but if it is left as it is without cooling, the solution system separates into two layers. Specifically, it separates into two layers, an equilibrium solution containing only a small amount of elastin molecules (upper layer) and a viscous coacervate layer (lower layer) in which the molecules are concentrated. This layer separation process is also reversible, and when the temperature is lowered to 25 ° C. or lower, the original transparent uniform solution is restored. This reversible self-assembly / dissociation property is "coacervation," which is an important property for normal elastin fibrosis and the development of elastic function.

The primary structural feature of tropoelastin, a precursor of elastin, is that hydrophobic regions rich in hydrophobic amino acids and cross-linked regions involved in intermolecular cross-linking are alternately repeated. There are repeating sequences of various hydrophobic amino acids in the hydrophobic region, and one of the repeating sequences is Val-Pro-Gly-Val-Gly (hereinafter, may be abbreviated as "VPGVG"; In the book, the amino acid sequence is described from left to right (the same applies hereinafter) from the N-terminal side to the C-terminal side), and the pentapeptide sequence is almost all animal species reported so far. Exists in. In addition, the synthetic polypeptide (VPGVG) _n (n ≧ 40) having this repeating sequence exhibits core selvation characteristics, suggesting that the VPPGVG repeating sequence is a sequence responsible for the elastic function of elastin.

As described above, the polypeptide having the repeating amino acid sequence of VPGVG has high utility value as a basic material such as a biomaterial and a material for a drug delivery system (DDS) because it has a self-assembling ability. However, in order to industrially utilize the polypeptide, it is necessary to synthesize a high molecular weight peptide having a very large number of repetitions (n) in the repetition sequence and which is difficult to prepare, which is time consuming and costly. For this reason, it has hardly been used industrially so far. As a report from overseas, it is possible to use a polymer having a genetically engineered Ile-Pro-Gly-Val-Gly (hereinafter, sometimes abbreviated as "IPGVG") repeating sequence as a biomaterial. It was previously proposed by Professor Dan W. Urry (see Non-Patent Document 1).

Here, the present inventors assume that the N-terminal amino acids (valine, isoleucine) are replaced with phenylalanine (Phe) in the conventionally reported synthetic polypeptides (polypentapeptide; (VPGVG) _n and (IPGVG) _n). We have found that the core selvation (self-assembly) ability can be expressed even with a low molecular weight that is easy to prepare because the number of repetitions (n) of the repeating sequence is small, and we have applied for a patent (Patent Document 1).

Japanese Unexamined Patent Publication No. 2012-126713

J. Phys. Chem. B, 101, 11007-11028 (1997)

As described above, _(VPGVG) poly pentapeptides the N-terminal amino acid is replaced with phenylalanine (Phe) of _{n ((FPGVG) n)} coacervation even low molecular weight (less number of repetitions) (self-assembly) ability exhibited about However, for other polypeptide sequences and peptides with shorter basic repeat sequences such as polytetrapeptides, whether or not the core selvation ability at a low molecular weight is exhibited. The current situation is that it is not known. If a peptide capable of self-assembly even with a lower molecular weight is developed, it is very advantageous for industrialization in various industries. Therefore, the development of such a new peptide is desired.

Therefore, an object of the present invention is to provide a novel peptide capable of expressing core selvation (self-assembly) ability even with a low molecular weight.

The present inventors have conducted diligent studies in view of the above-mentioned problems. In the process, it was found that the above-mentioned problem can be solved by a peptide having a structure represented by the following chemical formula 1, and the present invention has been completed.

That is, according to one form (first form) of the present invention, the following peptides (a) or (b) (provided that X ₁ -Pro-X _2- X _3- X ₄ is Val-Pro-Gly- (Except those that are Val-Gly or Phe-Pro-Gly-Val-Gly) are provided. _{In the present specification, "X 1-} Pro-X _2- X _3- X ₄ ", which is a repeating sequence in the following chemical formula 1, is designated as SEQ ID NO: 1.

(A) The following chemical formula 1:

During the ceremony
n is an integer from 3 to 300
X ₁ is tryptophan, tyrosine, valine, phenylalanine or cyclohexylalanine,
X ₂ is glycine or alanine,
X ₃ is a valine, phenylalanine, leucine, isoleucine or amino acid deletion.
X ₄ is, glycine, alanine, or amino acid deletions,
However, it is not _{that X 3} and _{X 4} are both an amino acid deletion,
Peptide represented by;
(B) A peptide obtained by deleting, substituting, and / or adding one or several amino acids to the peptide of (a), which exhibits temperature-dependent self-assembly.

Further, in the preferred form of the above-mentioned form, n is an integer of 3 to 10, and in a more preferable form, X ₁ is tryptophan, cyclohexylalanine, or phenylalanine, X ₂ is glycine, and X ₃ is valine. , X ₄ is a glycine or an amino acid deletion.

In a more preferred form, the peptide preferably has a repeating sequence represented by any of the amino acid sequences of SEQ ID NOs: 2 to SEQ ID NO: 12 , one of the amino acids of SEQ ID NO: 2, SEQ ID NO: 8 and SEQ ID NO: 9. It is more preferable to have a repeating sequence represented by the sequence, and it is particularly preferable that the sequence consists of any of the amino acid sequences of SEQ ID NO: 14 to SEQ ID NO: 36 , SEQ ID NO: 19 , SEQ ID NO: 20 , SEQ ID NO: 21 , and sequence. It is particularly preferably composed of any of the amino acid sequences of No. 24 , SEQ ID NO: 25 , SEQ ID NO: 30 , and SEQ ID NO: 33.

Further, according to another form (second form) of the present invention, two peptides selected from the group consisting of the peptide of the following (d) and the peptide of the following (e) are -Cys-S-S-Cys. -Peptides linked via groups are also provided:
(D) The following chemical formula 2:

During the ceremony
n is an integer from 3 to 300
X ₅ is tryptophan, tyrosine, valine, isoleucine, phenylalanine or cyclohexylalanine.
X ₆ is glycine or alanine,
X ₇ is a valine, phenylalanine, leucine, isoleucine or amino acid deletion.
X ₈ is a glycine, alanine or amino acid deletion and
However, it is not _{that X 7} and _{X 8} are both the amino acid deletion,
Peptide represented by;
(E) A peptide obtained by deleting, substituting, and / or adding one or several amino acids to the peptide of (d), which exhibits temperature-dependent self-assembly.

Furthermore, according to still another embodiment of the present invention, there is provided a method of self-assembling peptides, comprising the step of heating an aqueous solution in which the peptides are dissolved.

Further, according to still another embodiment of the present invention, a peptide assembly is provided in which the peptides are self-assembled.

Furthermore, according to still another form of the present invention, a cell culture substrate containing the peptide or the peptide aggregate, and a method for producing a cell sheet using the same are also provided.

According to the present invention, it is possible to provide a novel peptide capable of expressing core selvation (self-assembly) ability even at a low molecular weight.

It is explanatory drawing for demonstrating the core selvation property of elastin. It is explanatory drawing for demonstrating the state of the chemical synthesis of the peptide by the solid phase method in an Example. It is explanatory drawing for demonstrating the state of the chemical synthesis of the peptide by the solid phase method in an Example. It is a graph which shows the result of having evaluated the core selvation property of the peptide (WPGVG) _{3 synthesized in an Example.} It is a graph which shows the result of having evaluated the core _{selvation property of (YPGVG) 5} , which is a peptide synthesized in an Example. It is a graph which shows the result of having evaluated the core _{selvation property of (VPGFG) 4} , which is a peptide synthesized in an Example. It is a graph which shows the result of having evaluated the core _{selvation property of (FPGVA) 4} , which is a peptide synthesized in an Example. It is a graph which shows the result of having evaluated the core _{selvation property of (FPAVG) 4} , which is a peptide synthesized in an Example. It is a graph which shows the result of having evaluated the core selvation property of the peptide (FPGFG) _{4 synthesized in an Example.} It is a graph which shows the result of having evaluated the core selvation property of the peptide (ChaPGVG) _{3 synthesized in an Example.} It is a graph which shows the result of having evaluated the core _{selvation property of (FPGV) 3} , which is a peptide synthesized in an Example. It is a graph which shows the result of having evaluated the core _{selvation property of (FPGG) 6} which is the peptide synthesized in an Example. It is a graph which shows the result of having evaluated the core evaluation property of the peptide (FPGLG) _{5 synthesized in an Example.} It is a graph which shows the result of having evaluated the core evaluation property of the peptide (FPGIG) _{5 synthesized in an Example.} It is a graph which shows the result of having evaluated the core selvation property of N-dimer which is a peptide dimer synthesized in an Example. It is a graph which shows the result of having evaluated the core selvation property of C-dimer which is a peptide dimer synthesized in an Example. It is a graph which shows the result of having evaluated the core selvation property of the N-monomer which is a synthetic intermediate of the peptide dimer (N-dimer) synthesized in an Example. It is a graph which shows the result of having evaluated the core selvation property of the C-monomer which is a synthetic intermediate of the peptide dimer (C-dimer) synthesized in an Example. The results of evaluating the core selvation characteristics of the peptide dimers (N-dimer and C-dimer) synthesized in the examples are the corresponding synthetic intermediate monomers (N-monomer and C-monomer) and their synthetic raw materials. (FPGVG) ₅ is a graph shown in comparison with 5.

Hereinafter, specific embodiments for carrying out the present invention will be described in detail, but the technical scope of the present invention is not limited to the following specific embodiments.

According to one embodiment (first embodiment) of the present invention, the following peptides (a) or (b) are provided.

(A) The following chemical formula 1:

During the ceremony
n is an integer from 3 to 300
X ₁ is tryptophan, tyrosine, valine, phenylalanine or cyclohexylalanine,
X ₂ is glycine or alanine,
X ₃ is a valine, phenylalanine, leucine, isoleucine or amino acid deletion.
X ₄ is, glycine, alanine, or amino acid deletions,
However, it is not _{that X 3} and _{X 4} are both an amino acid deletion,
Peptide represented by;
(B) A peptide obtained by deleting, substituting, and / or adding one or several amino acids to the peptide of (a), which exhibits temperature-dependent self-assembly.

Here, from the range of peptides according to the present invention, X ₁ -Pro-X _2- X _3- X ₄ is Val-Pro-Gly-Val-Gly, and X ₁ -Pro-X _2- Those in which X _3- X ₄ is Ph-Pro-Gly-Val-Gly are excluded.

In X _1- Pro-X _2- X _3- X ₄ , X ₃ and X ₄ can be amino acid deletions (ie, amino acids are absent), _{but both X 3} and X ₄ are amino acid deletions. There is no. Therefore, the repeating unit (X _1- Pro-X _2- X _3- X ₄ ) of the peptide according to the present invention is 4 amino acids or 5 amino acids. Then, n in the peptide (peptide represented by Chemical Formula 1) of (a) represents the number of repetitions (degree of polymerization) of 4 amino acids or 5 amino acids which are the repeating units. In the present invention, this n is indispensable to be an integer of 3 or more. This is because the peptide expresses the core selvation (self-assembly) ability only when n ≧ 3. On the other hand, regarding the upper limit, n is an integer of 300 or less as a molecular size that can be treated proteinically. Here, with respect to the lower limit, n is preferably an integer of 4 or more, more preferably an integer of 5 or more, and even more preferably an integer of 6 or more. On the other hand, regarding the upper limit, n is preferably an integer of 100 or less, and more preferably an integer of 10 or less.

As an example of a preferred embodiment of the peptide of (a) (peptide represented by Chemical Formula 1), those having the repeating sequence of any one of the following SEQ ID NOs: 2 to 12 can be mentioned.

-WPGVG (SEQ ID NO: 2)
-YPGVG (SEQ ID NO: 3)
VPGFG (SEQ ID NO: 4)
FPGVA (SEQ ID NO: 5)
・ FPAVG (SEQ ID NO: 6)
FPGFG (SEQ ID NO: 7)
-ChaPGVG (SEQ ID NO: 8)
FPGV (SEQ ID NO: 9)
FPGG (SEQ ID NO: 10)
FPGLG (SEQ ID NO: 11)
・ FPGIG (SEQ ID NO: 12)
Here, the cyclohexyl alanine (Cha), an amino acid having a methyl group bonded to the asymmetric carbon atom of the alanine _{(NH 2 -CH (CH 3)} -COOH) is substituted by a cyclohexyl group.

Examples of the peptide according to the present invention in which WPGVG (SEQ ID NO: 2) is a repeating sequence include n = 2 (SEQ ID NO: 14) and n = 3 (SEQ ID NO: 15). Examples of the peptide according to the present invention in which YPGVG (SEQ ID NO: 3) is a repeating sequence include n = 3 (SEQ ID NO: 16), n = 4 (SEQ ID NO: 17), and n = 5 (SEQ ID NO: 18). is there. Examples of the peptide according to the present invention in which VPGFG (SEQ ID NO: 4) is a repeating sequence include n = 4 (SEQ ID NO: 19), n = 5 (SEQ ID NO: 20), and n = 6 (SEQ ID NO: 21). is there. Examples of the peptide according to the present invention in which FPGVA (SEQ ID NO: 5) is a repeating sequence include n = 3 (SEQ ID NO: 22), n = 4 (SEQ ID NO: 23), n = 5 (SEQ ID NO: 24), n =. There is one of 6 (SEQ ID NO: 25). An example of a peptide according to the present invention in which FPAVG (SEQ ID NO: 6) is a repeating sequence is n = 4 (SEQ ID NO: 26). Examples of the peptide according to the present invention in which FPGFG (SEQ ID NO: 7) is a repeating sequence include n = 3 (SEQ ID NO: 27) and n = 4 (SEQ ID NO: 28). Examples of the peptide according to the present invention in which ChaPGVG (SEQ ID NO: 8) is a repeating sequence include n = 3 (SEQ ID NO: 29) and n = 4 (SEQ ID NO: 30). Examples of the peptide according to the present invention in which FPGV (SEQ ID NO: 9) is a repeating sequence include n = 3 (SEQ ID NO: 31), n = 4 (SEQ ID NO: 32), and n = 5 (SEQ ID NO: 33). is there. An example of a peptide according to the present invention in which FPGG (SEQ ID NO: 10) is a repeating sequence is n = 6 (SEQ ID NO: 34). An example of a peptide according to the present invention in which FPGLG (SEQ ID NO: 11) is a repeating sequence is n = 4 (SEQ ID NO: 35). An example of a peptide according to the present invention in which FPGIG (SEQ ID NO: 12) is a repeating sequence is n = 4 (SEQ ID NO: 36).

Among these, peptides having WPGVG (SEQ ID NO: 2), ChaPGVG (SEQ ID NO: 8), or FPGV (SEQ ID NO: 9) as a repeating sequence are used to exhibit core selvation (self-assembly) ability at a lower molecular weight. preferable. Peptides having these repeating sequences exhibit core selvation (self-assembly) even at a very small molecular weight of n = 3. In particular, when FPGV (SEQ ID NO: 9), which is a tetrapeptide, is used as a repeating sequence, core selvation (self-assembly) is exhibited with only 12 amino acids when n = 3, so when considering industrialization or the like. Can be said to be an extremely useful peptide. To generalize the fact that these repeating sequences are preferable, in the chemical formula 1, X ₁ is preferably tryptophan (W), cyclohexylalanine (Cha) or phenylalanine (Phe), and X ₂ is glycine (Gly). X ₃ is preferably valine (Val), X ₄ is preferably glycine (Gly) or amino acid deletion, and more preferably glycine (Gly). On the other hand, X ₁ is preferably not tyrosine and X ₄ is preferably not alanine (Ala). In particular, the peptides provided by the present invention are designed based on the amino acid sequence of elastin. For this reason, the amino acid sequences located in the center of the repetitive sequence _{(X 1 -Pro-X 2 -X} 3 -X 4) _{"Pro-X 2"} are conserved similar to elastin as "Pro-Gly" (That is, X ₂ is Gly) is preferable. According to such a form, it is possible to exert a temperature-dependent self-assembly ability.

Further, when the peptide according to the present invention is compared as the peptide of the embodiment, it is excellent in self-assembling ability (that is, a coacervation phenomenon occurs even at a low concentration of 10 mg / ml) (VPGFG). ₄ (SEQ ID NO: 19 ), (VPGFG) ₅ (SEQ ID NO: 20 ), (VPGFG) ₆ (SEQ ID NO: 21 ), (FPGVA) ₅ (SEQ ID NO: 24 ), (FPGVA) ₆ (SEQ ID NO: 25 ), (ChaPGVG) ₄ (SEQ ID NO: 30 ) and (FPGV) ₅ (SEQ ID NO: 33 ) are preferred. Further, (VPGFG) ₅ (SEQ ID NO: 20 ), (VPGFG) ₆ (SEQ ID NO: 21 ), (ChaPGVG) ₄ (SEQ ID NO: 30 ), and (FPGV) ₅ (SEQ ID NO: 33 ) are more preferable, and (ChaPGVG) ₄ (SEQ ID NO: 30 ) is particularly preferred.

Incidentally, C-terminal, the peptides provided by the present invention, a carboxyl group (-COOH), a carboxylate (-COO ^-), may be any of an amide (-CONH ₂₎ or an ester (-COOR). Here, when the C-terminal is an ester (-COOR), the R is, for example, a C _{1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, or C 3} such as cyclopentyl or cyclohexyl. _{-8 cycloalkyl group, C 6-12} aryl group such as phenyl, α-naphthyl _{, phenyl-C 1-2} alkyl group such as benzyl, phenethyl or α-naphthyl-C _1-2 alkyl group such as α-naphthylmethyl _{C 7-14} aralkyl groups such as pivaloyloxymethyl ^group, -OBu ^t, -OPac (phenacyl group), - OTCE (trichloroethyl group), - ONb (p-nitrobenzyl group), - ODpm (diphenylmethyl Group), -OBzl (OMe) (p-methoxybenzyl group), -OPic (4-picoryl group) and the like.

Further, the N-terminal amino group of the peptide provided by the present invention is a protecting group (for example, _{C 1-6 acyl group such as C 1-6} alkanoyl such as formyl group and acetyl group, Fmoc group, Boc group and the like). It may be protected by.

Furthermore, the peptides provided by the present invention may be free or salts. As such salts, salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts) are used, and in particular, physiologically acceptable acid addition salts. Is preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, trifluoroacetic acid) and the like are used.

According to the present invention, in addition to the peptide represented by Chemical Formula 1 shown in (a) above, a peptide having an amino acid sequence in which the amino acid sequence of the peptide is slightly modified also has a temperature-dependent self-assembly ability. As long as it has, it can be included in the technical scope of the present invention. Specifically, as described in (b) above, in the peptide of (a), one or several (preferably 1 to 8, more preferably 1 to 4, still more preferably 1 to 2). Peptides in which amino acids are deleted, substituted, and / or added can also be included in the technical scope of the present invention as long as they have a temperature-dependent self-assembling ability. Whether or not a specific peptide exhibits temperature-dependent self-assembly ability can be determined by the method described in Examples described later.

Furthermore, amino acids have D-type and L-type optical isomers. Examples of using L-type amino acids are described in the examples described later, but peptides in which at least a part of them are replaced with D-type amino acids are also considered to exhibit similar self-assembly properties, and the present application. It can be included in the technical scope of the invention. That is, the amino acids constituting the peptide of the present invention represented by Chemical Formula 1 may be L-type or D-type at all sites, but preferably all amino acids are L-type.

According to another form (second form) of the present invention, a dimer of a peptide oligomer (also referred to herein as a "peptide dimer") consisting of a repeating sequence of tetrapeptides or pentapeptides as described above is also available. It is also provided. Specifically, according to another embodiment of the present invention, two peptides selected from the group consisting of the peptide (d) below and the peptide (e) below form a -Cys-S-S-Cys- group. Peptides linked through are also provided:
(D) The following chemical formula 2:

During the ceremony
n is an integer from 3 to 300
X ₅ is tryptophan, tyrosine, valine, isoleucine, phenylalanine or cyclohexylalanine.
X ₆ is glycine or alanine,
X ₇ is a valine, phenylalanine, leucine, isoleucine or amino acid deletion.
X ₈ is a glycine, alanine or amino acid deletion and
However, it is not _{that X 7} and _{X 8} are both the amino acid deletion,
Peptide represented by;
(E) A peptide obtained by deleting, substituting, and / or adding one or several amino acids to the peptide of (d), which exhibits temperature-dependent self-assembly.

Here, the peptide of (d) above, except that X ₅ may be a isoleucine, is the same as the peptide of (a) in the first embodiment of the present invention described above. Further, from the range of the peptide of (d) and the peptide of (e) constituting the peptide according to the present form (second form), "X _{1" which was excluded from the above-mentioned first form of the present invention.} -Pro-X _2- X _3- X ₄ is Val-Pro-Gly-Val-Gly or Phe-Pro-Gly-Val-Gly "is not excluded.

In the peptide dimer according to this embodiment (second embodiment), two peptides selected from the group consisting of the peptide of (d) and the peptide of (e) above are via a -Cys-S-S-Cys- group. It is connected by. Here, "two peptides are linked via a -Cys-S-S-Cys- group (a structure in which S atoms existing in the side chains of two Cys residues are disulfide bonds (-S-S-)). There is no particular limitation on the specific form to be used. For example, the thiol bond (-SH) of the Cys residue of "N-monomer (the N-terminal of the peptide to which the cysteine (Cys) residue is bound)" described in Example 2 described later is disulfide-bonded. (N-dimer) in which a -Cys-S-S-Cys- group is formed by Similarly, the thiol bond (-SH) of the Cys residue of the "C-monomer (the C-terminal of the peptide to which the cysteine (Cys) residue is bound)" described in Example 2 described later is disulfide-bonded. As a result, the -Cys-S-S-Cys- group may be formed (C-dimer). Further, a -Cys-S-S-Cys- group is formed by a disulfide bond of the thiol bond (-SH) of each Cys residue of the N-monomer and the C-monomer (heterodimer). May be good.

The method for producing the peptide provided by the present invention is not particularly limited, and conventionally known findings regarding the acquisition of the peptide can be appropriately referred to. For example, the peptides of the invention can be produced according to known peptide synthesis methods.

The molecular weight of the peptide according to the present invention (the peptide according to the first form and the peptide dimer according to the second form) is not particularly limited, but the reason why a lower molecular weight is preferable in terms of industrial production and the like. Therefore, it is preferably less than 3000, more preferably 2500 or less, and further preferably 2000 or less.

The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. That is, the target peptide can be produced by condensing the amino acids constituting the peptide of the present invention with the residual portion and removing the protecting group when the product has a protecting group. Examples of known condensation methods and desorption of protecting groups include the methods described in (i) to (v) below.
(I) M. Bondanzaky and M.D. A. Ondetti, Peptide Synthesis, Interscience Publics, New York (1966)
(Ii) Schroeder and Loubke, The Peptide, Academic Press, New York (1965)
(Iii) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
(Iv) Haruaki Yajima and Shunpei Sakakibara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977)
(V) Supervised by Haruaki Yajima, Development of Continued Drugs, Volume 14, Peptide Synthesis, Hirokawa Shoten The peptide thus obtained can be purified and isolated by a known purification method. Here, examples of the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like.

When the peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method or a method similar thereto, and conversely, when the peptide is obtained by a salt, it can be converted into a known method or a method thereof. It can be converted to a free form or other salt by a similar method.

A commercially available peptide synthesis resin can be usually used for the synthesis of the peptide of the present invention. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxymethylmethyl. Examples thereof include phenylacetamide methyl resin, polyacrylamide resin, 4- (2', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2', 4'-dimethoxyphenyl-Fmoc aminoethyl) phenoxy resin and the like. Using such a resin, amino acids appropriately protected with an α-amino group and a side chain functional group are condensed on the resin according to the sequence of the target peptide and according to various condensation methods known per se. At the end of the reaction, the peptide is excised from the resin and at the same time various protecting groups are removed to obtain the desired peptide.

Regarding the condensation of the above-mentioned protected amino acids, various activation reagents that can be used for peptide synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N'-diisopropylcarbodiimide, N-ethyl-N'-(3-dimethylaminoprolyl) carbodiimide and the like are used. It is also possible to use a condensing agent such as TBTU, HBTU, HATU, BOP, PyBOP. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (for example, HOBt, HOOBt, HOAt), or the protected amino acid is activated in advance as a symmetric acid anhydride, a HOBt ester, or a HOOBt ester. After that, it can be added to the resin.

As the solvent used for activating the protected amino acid and condensing with the resin, a solvent known to be used for the peptide condensation reaction can be appropriately selected. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide and the like. Sulfoxides, ethers such as pyridine, dioxane, tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or suitable mixtures thereof, etc. are used. The reaction temperature is appropriately selected from the range known to be usable for the peptide bond forming reaction, and is usually in the range of about -20 to about 50 ° C. Activated amino acid derivatives are usually used in excess of 1.1-10 fold. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation is not obtained even after repeating the reaction, the unreacted amino acid can be acetylated with acetic anhydride or acetylimidazole so as not to affect the subsequent reaction.

The protection of functional groups that should not be involved in the reaction of the raw material, the protecting groups, the elimination of the protecting groups, the activation of the functional groups involved in the reaction, and the like can be appropriately selected from known groups or known means.

Examples of the protecting group for the amino group of the raw material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl and trifluoroacetyl. , Phtaloyl, formyl, 2-nitrophenylsulphenyl, diphenylphosphinoti oil, Fmoc and the like are used.

The carboxyl group can be, for example, linear, branched or such as alkyl esterified (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, trichloroethyl, etc. Cyclic, substituted or unsubstituted alkyl esterification, aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl ester, diphenylmethyl ester, 4- It can be protected by picoryl ester), phenacyl esterification, benzyloxycarbonyl hydrazide, t-butoxycarbonyl hydrazide, trityl hydrazide and the like.

Protecting group removal (desorption) methods include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen anhydride, methanesulfonic acid, trifluo. Acid treatment with lomethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine and the like, reduction with sodium in liquid ammonia and the like are also used. The desorption reaction by the acid treatment is generally carried out at a temperature of about -20 ° C to about 40 ° C, but in the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethylsulfide, 1, Addition of cation trapping agents such as 4-butanedithiol, 1,2-ethanedithiol and the like is effective. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indol protecting group of tryptophan is the above 1,2-ethanedithiol and 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkaline treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

Activated carboxyl groups of the raw material include, for example, the corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenols, 2,4,5-trichlorophenols, 2,4-dinitrophenols, Esters with cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccimid, N-hydroxyphthalimide, HOBt)] and the like are used. As the activated amino group of the raw material, for example, the corresponding phosphate amide is used.

As another method for obtaining a peptide, for example, first, the α-carboxyl group of the carboxy-terminal amino acid is amidated to protect it, then the peptide chain is extended to the amino group side to the desired chain length, and then the N of the peptide chain is obtained. A peptide from which only the protective group of the α-amino group at the terminal has been removed and a peptide from which only the protective group of the carboxyl group at the C terminal has been removed are produced, and these peptides are condensed in a mixed solvent as described above. The details of the condensation reaction are the same as above. After purifying the protected peptide obtained by condensation, all protecting groups can be removed by the above method to obtain a desired crude peptide. This crude peptide can be purified by making full use of various known purification means, and the main fraction can be freeze-dried to obtain an amide compound of the desired peptide.

To obtain an ester of a peptide, for example, the α-carboxyl group of the carboxy-terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then an ester of the desired peptide is obtained in the same manner as the amide of the peptide. be able to.

Furthermore, the peptide provided by the present invention can also be produced by culturing a transformant containing a polynucleotide encoding the same and separating and purifying the peptide from the obtained culture. The polynucleotide encoding the peptide of the present invention may be DNA, RNA, or a DNA / RNA chimera. DNA is preferably mentioned. In addition, the polynucleotide may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA or a hybrid of DNA: RNA. In the case of a single strand, it may be either a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).

Polynucleotides encoding the peptides of the invention include genomic DNA, genomic DNA libraries, mammals (eg, humans, cows, monkeys, horses, pigs, sheep, goats, dogs, cats, guinea pigs, rats, mice, rabbits). , Hamster, etc.) (eg, hepatocytes, splenocytes, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangium cells, Langerhans cells, epidermal cells, epithelial cells, cup cells, endothelial cells, smooth muscle cells) , Fibroblasts, fibrous cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B cells, natural killer cells, obese cells, neutrophils, basal spheres, eosinophils, monospheres), Giant nuclei, synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary or stromal cells, or precursor cells, stem cells or cancer cells of these cells] or any tissue in which they are present [For example, brain, brain parts (eg, olfactory bulb, tonsillar nucleus, basal sphere, hippocampus, thorax, hypothalamus, cerebral cortex, spinal cord, cerebrum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad , Thyroid, bile sac, bone marrow, adrenal, skin, lung, gastrointestinal tract (eg colon, small intestine), blood vessels, heart, thoracic gland, spleen, submandibular gland, peripheral blood, prostate, testicles, ovary, placenta, uterus, bone, Examples include cDNA derived from joints, adipose tissue (eg, brown adipose tissue, white adipose tissue), skeletal muscle, etc., synthetic DNA, and the like. The genomic DNA and cDNA encoding the protein or its partial peptide used in the present invention use the genomic DNA fraction and the total RNA or mRNA fraction prepared from the above-mentioned cells / tissues as templates, respectively, and carry out a polymerase chain reaction (Polymerase Chain). It can also be amplified directly by the Reaction) (PCR method), the (Loop-Mediated Isothermal Amplification) LAMP method, and the RT-PCR method using reverse transcriptase.

Further, as described above, the peptide dimer according to the second form of the present invention has a cysteine (Cys) residue at the N-terminal or C-terminal of the peptide or the like according to the first form of the present invention as a synthetic intermediate thereof. ("N-monomer" and "C-monomer" described in Example 2) are used. The peptide synthesis method according to the first aspect of the present invention described above can be similarly applied to these monomers.

Then, the peptide dimer according to the second embodiment of the present invention can be obtained by forming a disulfide bond between the cysteine (Cys) residues of the monomers such as the N-monomer and the C-monomer described above. The specific method and conditions for carrying out this disulfide bond formation reaction are not particularly limited, and conventionally known findings can be appropriately referred to. For example, as described in Example 2 described later, the above-mentioned disulfide bond formation (and peptide dimer) is formed by air-oxidizing an N-monomer or C-monomer in an aqueous ammonium carbonate solution at a temperature of about 1 to 10 ° C. Synthesis) is possible.

The peptides and peptide dimers provided by the present invention have the property of self-assembling in a temperature-dependent manner (temperature-dependent self-assembly). That is, when the temperature of the solution in which it is dissolved rises, it reversibly self-assembles at a certain point in time to form a condensate, and as it cools, the self-assembly is eliminated and the solution returns to the original dissolved state. Utilizing this property, according to another embodiment of the present invention, the peptides are self-assembled, which comprises the step of heating an aqueous solution in which the peptide according to the first or second embodiment of the present invention is dissolved. A method is provided. Further, according to still another embodiment of the present invention, there is provided a peptide assembly in which the peptides of the present invention described above are self-assembled.

Self-assembly of the peptides of the invention can be performed by heating the solution in which the peptides of the invention are dissolved.

The concentration of the peptide in the peptide solution is not particularly limited, but is preferably about 1 to 100 mg / ml. Further, the heating means is not particularly limited, and heating means such as a water bath, a block heater, and an incubator, which are generally used in the technical field, can be used in the same manner. The heating temperature is also not particularly limited as long as it is a temperature at which peptide core selvation (self-assembly) occurs. The lower the temperature at which peptide core selvation (self-assembly) occurs, the more preferable. As an example, when the turbidity is measured by the method described in Examples described later, the temperature of the solution having a turbidity of 1.0 is preferably 60 ° C. or lower, more preferably 50 ° C. or lower, and further. It is preferably 40 ° C. or lower, particularly preferably 30 ° C. or lower, and most preferably 25 ° C. or lower.

The peptides provided by the present invention are expected to be widely used as raw materials for biomaterials for tissue engineering, drug delivery systems (DDS), base materials for cosmetics, and immobilization materials for microorganisms and enzymes. ..

In addition, the peptides and aggregates thereof provided by the present invention are also useful as cell culture substrates (see JP-A-2012-126713). That is, according to still another embodiment of the present invention, a cell culture substrate containing the peptide or peptide aggregate according to the present invention is also provided.

In this case, the peptide provided by the present invention or an aggregate thereof may be used alone as a cell culture substrate, or may be used in combination with other conventionally known cell culture substrates. Other conventionally known cell culture substrates that can be used in combination with the peptides provided by the present invention and aggregates thereof include, for example, collagen, gelatin, chitin-chitosan, hyaluronic acid, fibronectin, laminin, proteoglycan, tenascin, and the like. Agarose, alginic acid, cellulose, hydroxyapatite, polylactic acid (PLA), polylactic acid glycol (PLGA), polyacrylic acid (PAAc), poly (2-hydroxyethyl methacrylate) (PHEMA), polycaprolactone, fluororesin (polytetrafluoro) (Ethethylene, etc.) and the like.

Furthermore, according to other forms of the invention, methods for producing cell sheets are also provided. Specifically, the method for producing a cell sheet provided by the present invention includes a step (culture step) of culturing cells using the cell culture substrate containing the peptide or peptide aggregate according to the present invention described above, and culturing. It is characterized in that it includes a step of recovering a cell sheet containing the cells (recovery step). As a result, a cell sheet containing the cultured cells is produced.

The specific method for carrying out the above-mentioned production method is not particularly limited, and conventionally known findings can be appropriately referred to. As an example of the culture method, for example, a method of culturing cells on a culture dish on which the cell culture substrate according to the present invention is spread is exemplified. According to this method, a two-dimensional tissue (cell sheet) is formed, and this can be easily separated and recovered. At this time, in order to form a two-dimensional tissue (cell sheet) and collect it, the cell culture substrate according to the present invention may be spread on a culture dish, and normal cell culture may be performed on the cell culture substrate. Then, after confirming that the cells have sufficiently grown, the cell sheet can be peeled off from the culture dish by adding a large amount of the culture solution and shaking the culture dish.

Since the cell culture substrate provided by the present invention is composed of the peptide according to the present invention or an aggregate thereof, it exhibits core selvation ability (temperature-dependent self-assembly). Therefore, when cells are cultured on the cell culture substrate according to the present invention and then the culture is cooled, the cell culture substrate is liquefied. Experiments have shown that even if the cultured cells are recovered after the cell culture substrate is liquefied in this way, the recovered cells retain the sheet-like shape.

That is, in a preferred embodiment of the method for producing a cell sheet described above, the recovery step includes a step of recovering the cell sheet after cooling the culture to liquefy the cell culture substrate. The temperature at which the culture is cooled is not particularly limited as long as the cell culture substrate can be liquefied, but as an example, it is preferable to cool the culture to 25 ° C. or lower, more preferably 15 ° C. or lower. , It becomes possible to sufficiently liquefy this by utilizing the core selvation ability of the cell culture substrate. On the other hand, the lower limit of the temperature at which the culture is cooled is not particularly limited, but the temperature is preferably 5 ° C. or higher from the viewpoint of allowing the cells to survive in a normal state.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to examples, but the technical scope of the present invention should not be construed as being limited to the following examples.

<< Example 1: Chemical synthesis of peptides >>
The peptides shown in Table 1 below were synthesized by a chemical synthesis method (solid phase method). Here, the Fmoc method was used as the chemical synthesis method (solid phase method). As the resin for peptide synthesis and the Fmoc amino acid, products manufactured by Watanabe Chemical Industry Co., Ltd. were used. As the synthetic reagent, products manufactured by Watanabe Chemical Industries, Ltd. and Wako Pure Chemical Industries, Ltd. were used.

Specifically, the target peptide was synthesized by sequentially binding the Fmoc amino acid to the synthetic resin (Fmoc-NH-SAL-Resin) (see FIGS. 2 and 3). Incidentally, C-terminal, in FIG amino group (-NH ₂₎ attached to (a terminal of a side of an amino acid A ₅₎ are those derived from synthesis resin used for the solid phase method, C synthesized by this An amide bond (-C (= O) -NH ₂ ) is present at the terminal (the same applies hereinafter). At this time, 0.45M HBTU-HOBt and 2M DIEA were used for the condensation of amino acids, TFA (adding a scavenger if necessary) was added to the resin in which the peptide had been introduced, and the mixture was stirred at 25 ° C. for 1 to 2 hours. , TFA was removed. The obtained residue was dissolved in a mixed solution of water and acetonitrile and then freeze-dried. The obtained peptide was purified by high performance liquid chromatography (HPLC, manufactured by Toso Co., Ltd.), and mass spectrometry by MALDI TOF-MS measurement (apparatus: Voyager-DE, manufactured by Applied Biosystems) was performed. It was confirmed that the peptide was obtained with high purity.

<< Example 2: Chemical synthesis of peptide dimer >>
Peptide dimers were synthesized by the following methods.

_{First, a peptide (C (FPGVG) 5} ; also referred to as "N-monomer") in which a cysteine (Cys) residue is introduced into the N-terminal of _{(FPGVG) 5} by the same chemical synthesis method (solid phase method) as described above and _{(FPGVG) 5} of the C-terminal peptide introduce a cysteine (Cys) residues _((FPGVG) 5 C; "C- monomer") were respectively synthesized. Subsequently, each of these N-monomers and C-monomers is air-oxidized according to the following scheme to form a disulfide bond to form an N-terminal crosslinked homodimer (also referred to as “N-dimer”) and C. Homo dimers (also referred to as "C-dimers") crosslinked at the terminals were synthesized respectively.

≪Measurement of core selvation (self-assembly) characteristics of peptides≫
For each of the peptides obtained above, aqueous peptide solutions (10 to 100 mg / ml) of various concentrations were prepared, and a spectrophotometer with a Perche temperature controller (JASCO Ubest V-560, manufactured by JASCO Corporation) was used. The turbidity was measured under a nitrogen stream. The measurement conditions were a measurement wavelength of 400 nm, a temperature range of 5 to 50 ° C., and a temperature change rate of 0.5 ° C./min. In this measurement, samples after HPLC purification were used for WPGVG concentrations of 20 mg / ml and 30 mg / ml. On the other hand, for other peptides, samples after crude purification by Sep-Pak (registered trademark) (Waters) were used.

The results are shown in Tables 2-11 below and FIGS. 4-14 below. In the table below, "○" indicates core selvation (self-assembly), "x" does not indicate core selvation, and "-" is unmeasured. ..

As shown in the above table and drawings, all of the peptides of the present invention exhibited sufficient core selvation (self-assembly) ability equivalent to that of conventionally known high molecular weight polypeptides.

≪Measurement of core selvation (self-assembly) characteristics of peptide dimers≫
For the peptide dimers (N-dimer and C-dimer) obtained above, an aqueous solution of 10 mg / ml was prepared, and the turbidity was measured by the same method and conditions as described above. In this measurement, a sample after HPLC purification was used. In addition, as a reference example, the turbidity of the N-monomer and C-monomer obtained above was measured in the same manner. The results are shown in FIGS. 15-18. Further, FIG. 19 shows a graph comparing the results of turbidity measurement for _{(FPGVG) 5,} which is a synthetic raw material of the above-mentioned monomer, including the results of turbidity measurement. As shown in FIG. 19, it can be seen that both the N-dimer and the C-dimer show core selvation at lower temperatures as compared to the corresponding monomers and have excellent self-assembling ability. As shown in FIG. 19, (FPGVG) ₅ did not show coacervation at a low concentration of 10 mg / ml.

From the above, it can be seen that according to the present invention, peptides and peptide dimers having coacervation (self-assembly) ability can be provided even with a low molecular weight. Furthermore, the peptides provided by the first embodiment of the present invention exhibit self-assembly start temperature near body temperature (37 ° C.) and are reversible, similar to high molecular weight polypeptides, despite their small molecular weight size. It was also confirmed that it shows the properties of general assembly and deassembly. It was also confirmed that the peptide dimer provided by the present invention self-assembles at a low temperature of 15 ° C. or lower even at a low concentration of 10 mg / ml, and exhibits reversible assembly and disassembly properties. It was.

The peptide provided by the present invention can be easily synthesized by the above-mentioned chemical synthesis method (of course, it may be genetically synthesized). In addition, since the length of the amino acid sequence is shorter than that of a conventionally known polypentapeptide, there is an advantage that the labor required for purification after synthesis can be significantly reduced.

Further, in the conventional solid-phase synthesis of a peptide, when each amino acid is bound to the resin as the solid phase, the introduction rate is not perfect at 95 to 99%, so that after the peptide is cut out from the resin. The content of the target substance is about 95 to 97%, and amino acids such as 1 residue and 4 residues are generally mixed as impurities. On the other hand, since the peptide provided by the present invention has a simple sequence structure, it is possible to use an amino acid that does not require a side chain protecting group in its synthesis. Therefore, if a completely single peptide can be synthesized by introducing each amino acid into the resin for synthesis in a yield of almost 100%, the peptide cut out from the resin without purification by HPLC. Can be used as it is. As described above, the peptide of the present invention, which has an advantage from the viewpoint of synthesis, is expected to be an extremely useful material in industry.

Specifically, the peptide provided by the present invention is water-soluble and can be used as various medical materials such that an efficient DDS carrier capable of containing a drug inside can be easily synthesized. It is expected that it can be done.

[SEQ ID NO: 1]
_{It is a repeating amino acid sequence (X 1-} Pro-X _2- X _3- X ₄ ) possessed by the peptide (a) in the present invention.
[SEQ ID NO: 2]
_{It is a repeating amino acid sequence when X 1} = W, X ₂ = G, X ₃ = V, X ₄ = G in the repeating amino acid sequence of SEQ ID NO: 1 used for peptide synthesis in Examples (the present invention). One embodiment).
[SEQ ID NO: 3]
_{It is a repeating amino acid sequence when X 1} = Y, X ₂ = G, X ₃ = V, X ₄ = G in the repeating amino acid sequence of SEQ ID NO: 1 used for peptide synthesis in Examples (the present invention). One embodiment).
[SEQ ID NO: 4]
_{It is a repeating amino acid sequence when X 1} = V, X ₂ = G, X ₃ = F, X ₄ = G in the repeating amino acid sequence of SEQ ID NO: 1 used for peptide synthesis in Examples (the present invention). One embodiment).
[SEQ ID NO: 5]
_{It is a repeating amino acid sequence when X 1} = F, X ₂ = G, X ₃ = V, X ₄ = A in the repeating amino acid sequence of SEQ ID NO: 1 used for peptide synthesis in Examples (the present invention). One embodiment).
[SEQ ID NO: 6]
_{It is a repeating amino acid sequence when X 1} = F, X ₂ = A, X ₃ = V, X ₄ = G in the repeating amino acid sequence of SEQ ID NO: 1 used for peptide synthesis in Examples (the present invention). One embodiment).
[SEQ ID NO: 7]
_{It is a repeating amino acid sequence when X 1} = F, X ₂ = G, X ₃ = F, X ₄ = G in the repeating amino acid sequence of SEQ ID NO: 1 used for peptide synthesis in Examples (the present invention). One embodiment).
[SEQ ID NO: 8]
_{It is a repeating amino acid sequence when X 1} = Cha, X ₂ = G, X ₃ = V, X ₄ = G in the repeating amino acid sequence of SEQ ID NO: 1 used for peptide synthesis in Examples (the present invention). One embodiment).
[SEQ ID NO: 9]
_{It is a repeating amino acid sequence in the case where X 1} = F, X ₂ = G, X ₃ = V, X ₄ = amino acid deletion in the repeating amino acid sequence of SEQ ID NO: 1 used for the synthesis of the peptide in the example ( One embodiment of the present invention).
[SEQ ID NO: 10]
_{It is a repeating amino acid sequence when X 1} = F, X ₂ = G, X ₃ = amino acid deletion, and X ₄ = G in the repeating amino acid sequence of SEQ ID NO: 1 used for the synthesis of the peptide in the example ( One embodiment of the present invention).
[SEQ ID NO: 11]
_{It is a repeating amino acid sequence when X 1} = F, X ₂ = G, X ₃ = L, X ₄ = G in the repeating amino acid sequence of SEQ ID NO: 1 used for peptide synthesis in Examples (the present invention). One embodiment).
[SEQ ID NO: 12]
_{It is a repeating amino acid sequence when X 1} = F, X ₂ = G, X ₃ = I, X ₄ = G in the repeating amino acid sequence of SEQ ID NO: 1 used for peptide synthesis in Examples (the present invention). One embodiment).
[SEQ ID NO: 13]
_{It is a repeating amino acid sequence when X 1} = V, X ₂ = G, X ₃ = W, X ₄ = G in the repeating amino acid sequence of SEQ ID NO: 1 used for peptide synthesis in Examples (Comparative Example). ).
[SEQ ID NO: 14]
It is an amino acid sequence of the polypeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in the examples is WPGVG and n = 2.}
[SEQ ID NO: 15]
It is an amino acid sequence of the polypeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in the examples is WPGVG and n = 3.}
[SEQ ID NO: 16]
It is an amino acid sequence of the polypeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in Examples is YPGVG and n = 3.}
[SEQ ID NO: 17]
It is an amino acid sequence of the polypeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in Examples is YPGVG and n = 4.}
[SEQ ID NO: 18]
It is an amino acid sequence of the polypeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in Examples is YPGVG and n = 5.}
[SEQ ID NO: 19]
It is an amino acid sequence of the polypeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in Examples is VPGFG and n = 4.}
[SEQ ID NO: 20]
It is an amino acid sequence of the polypeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in Examples is VPGFG and n = 5.}
[SEQ ID NO: 21]
It is an amino acid sequence of the polypeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in Examples is VPGFG and n = 6.}
[SEQ ID NO: 22]
It is an amino acid sequence of the polypeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in Examples is FPGVA and n = 3.}
[SEQ ID NO: 23]
It is an amino acid sequence of the polypeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in the examples is FPGVA and n = 4.}
[SEQ ID NO: 24]
It is an amino acid sequence of the polypeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in Examples is FPGVA and n = 5.}
[SEQ ID NO: 25]
It is an amino acid sequence of the polypeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in the examples is FPGVA and n = 6.}
[SEQ ID NO: 26]
It is an amino acid sequence of the polypentapeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in the examples is FPAVG and n = 4.}
[SEQ ID NO: 27]
It is an amino acid sequence of the polypeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in Examples is FPGFG and n = 3.}
[SEQ ID NO: 28]
It is an amino acid sequence of the polypeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in Examples is FPGFG and n = 4.}
[SEQ ID NO: 29]
It is an amino acid sequence of the polypeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in the examples is ChaPGVG and n = 3.}
[SEQ ID NO: 30]
It is an amino acid sequence of the polypeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in the examples is ChaPGVG and n = 4.}
[SEQ ID NO: 31]
It is an amino acid sequence of the polytetrapeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in Examples is FPGV and n = 3.}
[SEQ ID NO: 32]
It is an amino acid sequence of the polytetrapeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in Examples is FPGV and n = 4.}
[SEQ ID NO: 33]
It is an amino acid sequence of the polytetrapeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in Examples is FPGV and n = 5.}
[SEQ ID NO: 34]
It is an amino acid sequence of the polytetrapeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in Examples is FPGG and n = 6.}
[SEQ ID NO: 35]
It is an amino acid sequence of the polytetrapeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in the examples is FPGLG and n = 4.}
[SEQ ID NO: 36]
It is an amino acid sequence of the polytetrapeptide according to the present invention in which the repeating amino acid sequence (X ₁ -Pro-X _2- X _3- X _{4) synthesized in the examples is FPGIG and n = 4.}
[SEQ ID NO: 37]
_{The repeating amino acid sequence (X 1} -Pro-X _2- X _3- X ₄ ) synthesized in the examples is the amino acid sequence of the polypentapeptide having VPGWG and n = 3 (comparative example).

Claims (14)

The peptide of (a) below, based on the amino acid sequence of elastin:
(A) The following chemical formula 1:

During the ceremony
n is an integer from 3 to 300
X ₁ is tryptophan, tyrosine, valine, phenylalanine or cyclohexylalanine,
X ₂ is glycine or alanine,
X ₃ is a valine, phenylalanine, leucine, isoleucine or amino acid deletion.
X ₄ is, glycine, alanine, or amino acid deletions,
However, it is not _{that X 3} and _{X 4} are both an amino acid deletion,
A peptide having a repeating sequence represented by any of the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 12 and exhibiting temperature-dependent self-assembly. The peptide according to claim 1, wherein n is an integer of 3 to 10. The peptide according to claim 1 or 2, wherein X _{2 is glycine.} 10. One of claims 1-3, wherein X ₁ is tryptophan, cyclohexylalanine, or phenylalanine, X ₂ is glycine, X ₃ is valine, and X ₄ is glycine or an amino acid deletion. Peptide. The peptide according to any one of claims 1 to 4, which has a repeating sequence represented by any one of the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 9. The peptide according to any one of claims 1 to 5, which comprises the amino acid sequence of any one of SEQ ID NO: 15, SEQ ID NO: 18 to SEQ ID NO: 21, SEQ ID NO: 23 to SEQ ID NO: 26, and SEQ ID NO: 28 to SEQ ID NO: 36. .. The invention according to any one of claims 1 to 6, which comprises any one of the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 30, and SEQ ID NO: 33. peptide. Based on the amino acid sequence of the elastin, it becomes linked via the two peptides -Cys-S-S-Cys- group selected from peptides or Ranaru the following group (d) peptides:
(D) The following chemical formula 2:

During the ceremony
n is an integer from 3 to 300
X ₅ is tryptophan, tyrosine, valine, isoleucine, phenylalanine or cyclohexylalanine.
X ₆ is glycine or alanine,
X ₇ is a valine, phenylalanine, leucine, isoleucine or amino acid deletion,
X ₈ is a glycine, alanine or amino acid deletion and
However, it is not _{that X 7} and _{X 8} are both the amino acid deletion,
A peptide having a repeating sequence represented by any of the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 12 and exhibiting temperature-dependent self-assembly. The peptide according to claim 8 , wherein X ₆ is glycine. The peptide according to any one of claims 1 to 9, which has a molecular weight of less than 3000. A method for self-assembling peptides, comprising the step of heating an aqueous solution in which the peptide according to any one of claims 1 to 10 is dissolved. A peptide assembly obtained by self-assembling the peptides according to any one of claims 1 to 10. A cell culture substrate comprising the peptide according to any one of claims 1 to 10 or the peptide aggregate according to claim 11. A culture step of culturing cells using the cell culture substrate according to claim 13,
A recovery step for collecting a cell sheet containing cultured cells, and
A method for producing a cell sheet, including.

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