WO2005056589A1

WO2005056589A1 - Peptide having its self-assembling property induced at given salt concentration and self-assembled matter therefrom

Info

Publication number: WO2005056589A1
Application number: PCT/JP2004/019140
Authority: WO
Inventors: Kei Kamino; Masahiro Nakano; Jian-Ren Shen; Satoru Kanai
Original assignee: Marine Biotechnology Institute Co., Ltd.; National University Corporation Okayama University; Pharma Design Inc.
Priority date: 2003-12-15
Filing date: 2004-12-15
Publication date: 2005-06-23
Also published as: JPWO2005056589A1

Abstract

Novel self-assembling peptides, in particular, a peptide that self-assembles by mechanism different from that of peptide capable of amphiphilicity-based self-assembly and a novel self-assembling material of peptide dependent upon salt concentration wherein physiological salt concentration is a salt concentration threshold value. There are provided self-assembling peptides represented by, based on recurring sequences of the amino acid sequence of 3rd adhesive protein of barnacle, consensus sequence (SEQ ID NO. 1), sequence having charge cluster EHSHDHHDDD added to the N-terminal side of SEQ ID NO. 1 (SEQ ID NO. 2), 5th recurring sequence (SEQ ID NO. 3), 6th recurring sequence (SEQ ID NO. 4) and SEQ ID NOS. 5 to 12.

Description

Specification

Peptides causing self-assembly at specific salt concentrations and their self-assembly

The present invention relates to a peptide having a self-assembling property. More specifically, the present invention relates to a peptide exhibiting self-assembly by increasing the salt concentration, and a method for preparing the same. The peptide of the present invention can be used as a material such as a biomaterial. Background art

Although some proteins and peptides that show self-assembly (also called self-association) such as dimers and multimers are known, it was not easy to modify proteins and peptides before. So it was not considered a subject of material engineering or engineering. However, with the development of genetic recombination technology and the advancement of peptide synthesizers, in recent years, for example, the application of self-assembly of biomolecules such as peptides to regenerative medicine and drug delivery has been studied. Attention has been drawn to its usefulness in the design and manufacture of nanotechnology.

As a self-assembly system using peptides, Aggel i and others have reported that short peptides are converted into nanotapes on long polymers with a water-insoluble solvent (Non-Patent Document 1: A. Aggel i, et al. (1997) Nature, Vol. 386, pp. 259-262) ₀ Polymer molecules having two different regions, a hydrophilic region and a hydrophobic region, are hydrophobic in an aqueous solution like a surfactant. It is known that self-assembly is caused by acting on each other so as to cause the ionic moieties to separate from the aqueous phase, and experiments using various simple peptides have been conducted (Non-Patent Document 2: S. et al. Vauthey, et al. (2002) Proc. Natl. Acad. Sci. USA, Vol. 99 pp. 5355-5360).

Peptide materials formed by salt-concentration-dependent self-assembly include, for example, amphipathic peptides (such as arginine-alanine-aspartic acid) in which hydrophilic and hydrophobic amino acids alternate alternately. Patent Document 3: TC. Holmes, et al. (2000)

Natl. Acad. Sci. USA, Vol. 97, pp. 6728-6733) and (Non-Patent Document 4: S. Zhang, et al. (2002) Cur. Opin. Chem. Biol., 6, 865-871) The

However, all self-assembling peptides provided to date have been peptides that utilize a amphiphilic base sheet structure.

Non-patent document 1: A. Aggel i, et al. (1997) Nature, Vol. 386, pp. 259-262 Non-patent document 2: S. Vauthey, et al. (2002) Proc. Natl. Acad. Sci. USA, Vol. 99 pp. 5355-5360

Non-Patent Document 3: TC. Holmes, et al. (2000) Proc. Natl. Acad. Sci. USA, Vol. 97, pp. 6728-6733.

Non-patent document 4: S. Zhang, et al. (2002) Cur. Op in. Chem. Biol., 6, 865-871 Disclosure of the invention

An object of the present invention is to provide a novel self-assembling peptidic material. In particular, an object of the present invention is to provide a self-assembling peptide different from a self-assembling peptide based on amphipathicity. It is another object of the present invention to provide a salt concentration-dependent novel self-assembling material having a physiological salt concentration as a threshold.

Another object of the present invention is to provide a novel self-assembling peptide and a novel peptide material having a salt concentration-dependent self-assembly having a physiological salt concentration as a threshold.

The present inventors have studied various materials for the development of a new self-assembling peptide, and as a result, have found that Fujibbo third adhesion protein (Japanese Patent Laid-Open No. 09-299089 or (Kamino, K. (2001) Biochem. J 356, 503-507). It was also found that a completely new type of self-assembling peptide can be provided by analyzing the amino acid sequence information of this protein (also referred to as Mrcp20k). The Fujitsubo third adhesion protein (Mrcp-20k) is a simple protein with a molecular weight of 20 kDa and an isoelectric point of 4, and is known to consist of 6 repeats by Cys alignment. It is a protein that contains few hydrophobic amino acids and is very rich in charged amino acids.

The present inventors aligned each repetitive sequence of the amino acid sequence of Fujibbo Third Adhesion Protein based on Cys, and optimized it visually, specifically, while checking the arrangement of Cys. Based on this, a consensus sequence (SEQ ID NO: 1) was created. SEQ ID NO: 1 In addition to the peptide represented by, a peptide represented by the sequence (SEQ ID NO: 2) with a charge cluster EHSHDHHDDD added to the N-terminal side of SEQ ID NO: 1 is represented by the fifth repeat sequence (SEQ ID NO: 3) And the peptide represented by the sixth repeat sequence (SEQ ID NO: 4) were chemically synthesized by the Boc method, purified by reverse phase chromatography, and lyophilized.

After dissolving them in an appropriate aqueous solution, an appropriate salt aqueous solution was added, and the resulting mixture was confirmed by light scattering measurement, atomic force microscope observation, circular dichroism polarization measurement, congoled staining, etc. The present invention has been completed by confirming the formation and structure thereof.

This description includes part or all of the contents as disclosed in the description and / or drawings of Japanese Patent Application No. 2003-417215, which is a priority document of the present application. Brief Description of Drawings

FIG. 1 shows the results of verifying the self-assembly properties of the peptides of SEQ ID NOs: 1 to 4 by changing the salt concentration by light scattering measurement. The horizontal axis represents the molar concentration of NaCl, and the vertical axis represents the apparent molecular weight (kDa) by light scattering measurement. 1 to 4 in the figure show the results when SEQ ID NOS: 1 to 4 are used as samples, respectively.

Figure 2 shows an image of a peptide assembly (self-assembly) by an atomic force microscope (AFM). Figure 3 is a photograph showing the formation of a film-like body self-assembled structure can be observed with the naked eye (peptide concentration 10 mg / ral) ₀

Fig. 4 AFM observation of self-assembly with pores using peptide of SEQ ID NO: 3 (pH8)

Fig. 5 Observation of self-assembly using peptide of SEQ ID NO: 3 by AFM (pH 6) Fig. 6 Measurement result of CD spectrum of peptide of SEQ ID NO: 3

Figure 7 AFM observation of self-assembly using a peptide in which Cys is replaced by Ser in SEQ ID NO: 3 (pH ⁸ )

Figure 8 Results of CD spectrum of peptide in which Cys of SEQ ID NO: 3 has been replaced with Ser Best mode for carrying out the invention The invention of the present application includes a novel self-assembling peptide based on a repeating structure, which is newly found in Fujibbo Third Adhesion Protein (Japanese Patent Application Laid-Open No. 09-299089). Specifically, the peptide represented by the amino acid sequence of SEQ ID NO: 3 (SKLPCNDEHPCYRKEGGVVSCDCK) or SEQ ID NO: 4 (KTITC NEDHPCYHSYEEDGVTKSDCDCE) and one to several amino acid sequences of SEQ ID NO: 3 or SEQ ID NO: 4, preferably Is a peptide in which 1 to 8, more preferably 1 to 4, and even more preferably 1 or 2 amino acids are deleted, substituted, and Z or added, and in an aqueous solution. Includes peptides that have self-assembly properties.

Furthermore, the present invention includes a novel self-assembling peptide consisting of a consensus sequence based on a repeating structure, which has been newly found in Fujibbo Third Adhesion Protein (Japanese Patent Laid-Open No. 09-299089). Specifically, the peptide represented by SEQ ID NO: 1 (EKIPC NEKHPCYRCDADTKCSCDCE) or 1 to several amino acid sequences in SEQ ID NO: 1, preferably 1 to 8, more preferably 1 to 4, More preferably, a peptide having deletion, substitution, Z or addition of one or two amino acids, which has self-assembly properties in an aqueous solution, is included.

The repeated structure of the protein is regarded as multiple alignment, and based on this, a profile HMM is created by the hmmbuild and hmmcalibrate programs, and the consensus sequence is determined from this profile HMM by the hmmemit program in the HMMER program package. You can also.

Specific examples of deletion, substitution, and addition to the amino acid sequence of SEQ ID NO: 1 include, for example, deletion of the third and fourth Cys (the 14th and 20th amino acids from the amino terminus). Mutations are included. (D and E), (N and Q), (A, G, T, and S), and (H, R, and K) in SEQ ID NO: 1 can be substituted in each (). Means this. It may also include a sequence in which the hydrophobic amino acids in SEQ ID NO: 1 are substituted for each other. Four Cys in SEQ ID NO: 1, except for the deficient Cys described above, are required for self-assembly. Pro before the second Cys is preferred. The next amino acid after the second Cys is preferably the (Bulky) amino acid (W, Y, R, H). The second

The next basic amino acid following Cys (the second amino residue downstream from the second Cys) is preferably the same as SEQ ID NO: 1. Between the first and second Cys Preferably, the number of amino acids is four. It is also desirable that the distance between the two rear Cys (on the carboxyl side) is 1 amino acid.

In the present invention, a sequence having a charge cluster can be added to a repetitive structure or a self-assembling peptide based on a consensus sequence thereof, to the third adhesion protein of Fujibo (Japanese Patent Laid-Open No. 09-299089). Specifically, HEED, KHD, EEDHKHHDH, KKH: HRK, EHSHDHHDDD can be mentioned, and preferably, EHSHDHHDDD can be used. Furthermore, specifically, a polypeptide in which EHSHDHHDDD is bound to the amino acid terminal side of the peptide represented by SEQ ID NO: 1 (SEQ ID NO: 2: EHSHDHHDDDEKIPCNEKHPCYRCDADTKCSCDCE) can be mentioned.

The following peptides are also peptides that can have self-assembly properties, and are included in the present invention.

SEQ ID NO: 5 (first repeat sequence of Mrcp20k)

HEEDGVCNSNAPCYHCDANGENCSCNCE

SEQ ID NO: 6 (second repeat sequence of Mrcp20k)

ELFDCEAKKPDGSYAHPCRRC image NICKCSCT

SEQ ID NO: 7 (third repeat sequence of rcp20k)

TAIPCNEDHPCHHCHEEDDGDTHCHCSCE

SEQ ID NO: 8 (the fourth repeat sequence of Mrcp20k)

EHSHDHHDDDTHGECTKKAPCWRCEYNADLKHDVCGCECS

SEQ ID NO: 9 (the first repeat sequence of Balcp20k (adhesion protein of shirojifujibo))

HKHHDHGKSCSKSHPCYHCHTDCECN

SEQ ID NO: 10 (second repeat sequence of Balcp20k)

NHHHDDCNRSHRCWHKVHGVVSGNCNCN

SEQ ID NO: 1 1 (The third repeat sequence of Bal cp20k)

NLLTPCNQKHPCWRRHGKKHGLHRKFHGNACNCD

SEQ ID NO: 1 2 (Fourth repeated sequence of Balcp20k)

DRLVCNAKHPCWHKHCDCFC

Furthermore, the present invention includes a repetition of a third adhesion protein of Fujibobo (JP-A-09-299089). Ri return structure or self-assembling peptide sequences, as well as cell adhesion to the self-assembling peptide based rather on repeating structure of adhesion proteins (No. ² 003_325604) of Shirosujifujibbo, motifs such as metal binding or antimicrobial sequences based on the consensus sequence Also included are functionalized self-assembling polypeptides prepared by linking sequences. For example, RGD (arginine-glycine-aspartic acid), which is a cell adhesion motif, can be bound to the N- or C-terminal of the peptide represented by any one of SEQ ID NOS: 1 to 12 above.

In the present invention, at least one or more amino acids selected from the group consisting of substitutions, additions, and deletions in the amino acid sequence of any one of SEQ ID NOs: 1 to 12 described above are substituted with one or several amino acids. A polypeptide represented by a modified polypeptide having the following mutation: a peptide exhibiting a salt concentration-dependent self-assembly property.

Further, the present invention includes a gene encoding the peptide represented by any one of SEQ ID NOS: 1 to 12, and one or several amino acids in the amino acid sequence represented by any of SEQ ID NOs: 1 to 12. A peptide represented by a deletion, substitution, and Z or an added amino acid sequence, which encodes a peptide that exhibits salt concentration-dependent self-assembly, and includes, for example, SEQ ID NO: 1. The gene having the sequence represented by 3 or 14 is specifically included, specifically, the sequence of base numbers 43 3 to 501 in the sequence of SEQ ID NO: 1 described in JP-A-09-299089 and the same SEQ ID NO: Also included are genes represented by the sequence of base numbers 502 to 582 of 1.

Further, the present invention includes a recombinant vector obtained by recombining the above-described gene, and a method for expressing the self-assembling peptide of the present invention using the recombinant vector.

In addition to the general chemical synthesis, the peptide of the present invention can also be obtained by preparing a recombinant using a general microorganism or cultured cells after chemically synthesizing the corresponding DNA.

As the chemical synthesis method, the t_Boc method and the Fmoc method can be used, and can also be synthesized using a peptide synthesizer. Production by the genetic recombination method includes, for example, using a known expression vector for the gene shown in SEQ ID NO: 13 or 14; for example, pET or pGEMEX when using Escherichia coli as a host, or mammalian cells as a host. When In this case, a method of expressing each of them by recombination with pCI can be used. The peptide disclosed in the present invention relates to a peptide self-assembling material having an amino acid sequence completely different from that of a conventionally known self-assembling peptide and formed by a different principle.

Furthermore, all of the peptide-based materials up to now have self-assembly properties even at low salt concentrations, but the peptides discovered by the present inventors show that self-assembly does not occur until a specific salt concentration. The present invention relates to a peptide in which self-assembly is rapidly exhibited when a salt concentration exceeds a specific salt concentration threshold to form a peptide aggregate. Preferably, self-assembly can be performed at a salt concentration of 0.5 M or more, and self-assembly can be performed even when the salt concentration exceeds 1 M. The self-assembly effect of salts is higher for monovalent salts than for divalent salts. As the salt to be used, a usual appropriate salt can be used. For example, NaCl, KC1, CaCl ₂ , and MgC ^ can be used.

Under these conditions, the peptides represented by SEQ ID NOs: 1 to 12 described above, for example, the peptides represented by SEQ ID NOs: 1 to 4, do not aggregate randomly but regularly self-assemble due to the salt effect. I do. Furthermore, one to several, preferably one to eight, more preferably one to four, and even more preferably one or two amino acids A peptide having deletion, substitution, and / or addition, which forms a self-assembly of peptides (peptide assembly) by exposing a peptide having self-assembly properties to a salt in an aqueous solution can do.

Further, as the peptide concentration that can be used, for example, a _{1 0 / ζ Μ~1 0 ιη §} Ζ m 1. Depending on the peptide concentration, a film can be formed. By selecting conditions, nanofibers or hydrogels can be used. Further, as a peptide sequence, a peptide represented by the amino acid sequence represented by SEQ ID NO: 1 to 12 in which one or several amino acids other than Cys are deleted, substituted, and Z or added. By reacting a peptide exhibiting a salt concentration-dependent self-assembly property with a salt under alkaline conditions, more preferably at pH 8, a self-assembly of peptides having regular pores can be formed. it can. As the salt to be used, an ordinary appropriate salt can be used. For example, NaCl, KC1, CaCl „MgCl ₂ can be used. Self-assembly of the polypeptide of the present invention can be carried out by bringing the polypeptide of the present invention into contact with an aqueous solution having a salt concentration in the above range by an appropriate means. Examples of the contacting method include a method of dropping the peptide of the present invention into an aqueous solution having a high salt concentration in a container having a specific shape, a method of discharging an aqueous peptide solution into an aqueous solution having a high salt concentration, and a method of contacting a container having a specific shape. And a method of dissolving a salt in an aqueous peptide solution. Thus, for example, even in the case of recombinant expression by a microorganism or the like, it can be produced in a medium having a salt concentration equal to or lower than the threshold, and can be immobilized immediately by simply adding a salt thereto. This includes, for example, the advantage that the startup process can be greatly simplified in immobilizing microorganisms during the production of a microbial reactor. In recombinant expression, by adding these sequences as tags to the target protein, it is dissolved during expression, but as a component technology for easily recovering only the target protein by raising the salt concentration to a threshold. By preliminarily immobilizing the sequence on a carrier or the like, it can be applied to a technique for retaining a recombinant having the same tag on a carrier. The peptide obtained by chemically synthesizing the chemically synthesized peptide and the DNA encoding the amino acid sequence and then recombinantly expressing the peptide in a microorganism or cultured cells is a biomaterial for tissue engineering and a drug delivery system. It is expected to be used in a wide range of applications as a base material for cosmetics and as a raw material for immobilizing materials such as microorganisms and enzymes. In addition, the construction principle is different from those known so far and is based on a new principle, and is applicable to bottom-up technology in nanotechnology.

Example 1

Simplification of Mrcp-20k repeat sequence

The repeat structure of Mrcp-20k protein was regarded as multiple 'alignment, and based on this, a profile HMM was created using the hmmbui Id and hmmcalibrate programs. From this profile HMM, a consensus sequence was determined by the hmmemit program in the 圆 MER program 'package to obtain a consensus sequence. The consensus sequence is shown in SEQ ID NO: 1.

Example 2

Synthesis of each peptide Using an ABI430A type fully automatic solid-phase synthesizer, four types of peptides represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 were synthesized by the Boc method. Condensation was performed using 4 equivalents of each Boc-amino acid to obtain a protected peptide resin. The resulting protected peptide resin is treated with anhydrous hydrogen fluoride / P-cresol (8: 2) at -2 to -5 ° C for 60 minutes, cut out from the resin and deprotected to obtain a crude peptide. Was. The resulting crude peptide using reverse phase HPLC column (0DS), 0. containing 1% TFA H ₂ 0

Purification was performed by gradient elution using a system of -CH ₃ CN. Fractions containing the target compound in high purity were collected and freeze-dried to obtain the target peptide.

Example 3

Confirmation of self-assembly by light scattering measurement

Light scattering was measured using Dyna Pro manufactured by PROTEIN SOLUTIONS. SEQ ID NO: 1

Ultrapure water equipment (Milli-Q SP. T0C.

(Millipore)) to prepare a stock solution. The NaCl, KC1, MgSO ₄ or CaCl ₂ with respect to stock solution of each pair flops tide was added to so that such each final concentration, was filtered (0. 2 μ ηι) measured after filtration. As a result, the molecular weight was below the detection limit at a salt concentration of 0.5 M or less, but a sharp increase in the molecular weight was observed under conditions of high salt concentration of 0.5 M or more close to seawater. Figure 1 shows the results when NaC1 was used as the salt. In the figure, the peptides of SEQ ID NO: 1, 2, 3, or 4 are represented by 1, 2, 3, or 4, respectively.

From the results of these experiments, it was clarified that each of the peptides had a salt concentration-dependent self-assembly activity at a salt concentration of 0.5 M as a threshold.

Example 4

Confirmation of peptide self-assembly structure by atomic force microscope (AFM)

AFM used Olympus NVB100. NaCl was added to the stock solution (100 μM) of each peptide prepared in Example 2 to a final concentration of 1M, and after stirring, allowed to stand for 10 minutes and dialyzed against pure water did. After recovery, a part of the solution was dropped onto Mica, and water droplets were removed by drying, and the measurement was performed in the air mode. The measurement was performed in Tapping mode, and each sample was scanned twice in the range of 5 μηιΧ 5 μιη.

As a result, it was determined whether a mass with a certain width developed for each peptide. An image was obtained. The results for SEQ ID NO: 4 are shown in FIG.

In addition, by adjusting the peptide concentration to 10 mg / ml, formation of a film-like body that could be visually confirmed directly was also observed. The results are shown in Figure 3.

Example 5

Preparation of peptide aggregates (self-assemblies) with pores

The peptide represented by SEQ ID NO: 3 was dissolved in 10 mM sodium phosphate buffer (pH 8) so that the peptide concentration became 100 μM, and the mixture was allowed to stand at room temperature in a polypropylene tube. A 1/4 volume of a 5 M NaCl solution was added to the peptide solution, mixed gently, and treated at room temperature for 10 minutes. This was dialyzed twice against 200 volumes of 10 mM sodium phosphate buffer (pH 8) to remove excess NaCl. Using this technique, we succeeded in creating a structure with pores. Fig. 4 shows the results of analyzing the structure using AFM in the same manner as in Example 4. This structure had pores that were not found when the peptide was dissolved in pure water and a salt was added. The size of the pores is between 50 and 500 nm and has a very regular structure. The peptide represented by SEQ ID NO: 3 was dissolved in 10 mM sodium phosphate buffer (pH 6) so that the peptide concentration became 100, and the mixture was allowed to stand at room temperature in a polypropylene tube. A 1/4 volume of a 5 M NaCl solution was added to the peptide solution, mixed gently, and treated at room temperature for 10 minutes. This was dialyzed twice against a 200-fold volume of 10 mM sodium phosphate buffer (pH 6 §) to remove excess NaCl. Figure 5 shows the result of analyzing the structure by AFM. No regular pores can be observed. The CD spectrum of the secondary structure of the peptide of SEQ ID NO: 3 in pure water and sodium phosphate buffer of pH 2 to 10 was measured. A stock solution (100 μM) of each peptide dissolved in ultrapure water prepared by ultrapure water equipment (Milli_Q SP. T0C. (Millipore)) was added to various pH buffers (pH 2-3 and pH2). (10 mM sodium phosphate buffer for 6-8, 10 mM sodium acetate buffer for pH 4-5, 10 mM sodium phosphate buffer for pH 9-10), and react at room temperature for 12 hours. After that, CD spectra were measured using a J725 type circular dichroism dispersometer manufactured by JASCO Corporation. Fig. 6 shows the results. It can be seen that the secondary structure is different between pH8-10 (alli) and pH2-6 (acid).

Next, in the sequence of SEQ ID NO: 3, all Cys were replaced with Ser, And the CD spectrum was measured. FIG. 7 shows the result of analyzing the structure by AFM under the same conditions as in SEQ ID NO: 3 described above. In addition, the CD spectrum showed the same results at pH 2 to 10 as those at pH 2 to 6 of SEQ ID NO: 3 described above. Fig. 8 shows the results. From the above, it is considered that regular pores are generated by reacting a peptide represented by a sequence containing Cys with a salt under alkaline conditions. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Industrial applicability

The present invention provides peptides, especially peptides that are self-assembling in a salt concentration threshold-dependent manner, and novel self-assemblies.

Here, “self-assembly” is one of the key technologies when constructing micro-sized (nano-scale) structures. Protein complexes that function extracellularly often have self-assembly properties, and their construction principles are useful for introducing new principles into nanotechnology.

The self-assembling peptide of the present invention can be used as a peptide material as it is or as a substrate for tissue engineering biomaterials, drug delivery systems, and cosmetics by introducing motif sequences such as cell adhesion, metal binding, and antimicrobial sequences. It is expected to be widely used as a tag for immobilization materials such as microorganisms and enzymes, as a tag for purification and immobilization of recombinant protein technology.

In addition, the construction principle is different from what is known so far, and is based on a new principle, and can be applied to the bottom-up technology in nanotechnology.

In addition, the self-assembly itself has the potential to be applied as biomaterial itself, which is a key material for surgical treatment and regenerative medicine, and as a cosmetic base. Sequence listing free text

SEQ ID NO: 1 is the consensus sequence of SEQ ID NOs: 3-8.

SEQ ID NO: 2 is a sequence obtained by adding a sequence having a charge cluster to SEQ ID NO: 1.

SEQ ID NOs: 3 to 8 are self-assembling peptides based on the fifth, sixth, first, second, third, and fourth repeat structures in the repeat structure of the akafudjibo adhesion protein, respectively.

SEQ ID NOS: 9 and 12 are self-assembling peptides based on the first, second, third, and fourth repeat structures in the repeat structure of the adhesion protein of Syrosdiphujibo.

SEQ ID NOs: 13 and 14 are examples of genes encoding SEQ ID NOs: 3 and 4, respectively.

Claims

The scope of the claims

1. Peptides of (a) or (b)

(a) a peptide represented by the amino acid sequence of SEQ ID NO: 1

(b) a peptide represented by an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added to the amino acid sequence of SEQ ID NO: 1, having a salt concentration-dependent self-assembly property. Indicating peptides.

2. (a) or (b) peptides

(a) a peptide represented by the amino acid sequence of SEQ ID NO: 2

(b) a peptide represented by the amino acid sequence of SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted, and / or added, and has a salt concentration-dependent self-assembly property. Peptide.

3. Peptides of (a) or (b)

(a) a peptide represented by the amino acid sequence of SEQ ID NO: 3

(b) a peptide represented by an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added to the amino acid sequence of SEQ ID NO: 3, and has a salt concentration-dependent self-assembly property. Peptide.

4. (a) or (b) peptides

(a) a peptide represented by the amino acid sequence of SEQ ID NO: 4

(b) a peptide represented by an amino acid sequence in which one or several amino acids are deleted, substituted, and Z or added to the amino acid sequence of SEQ ID NO: 4, and have a salt concentration-dependent self-assembly property. Indicating peptides.

5. (a) or (b) peptides

(a) a peptide represented by any one of SEQ ID NOs: 5 to 11;

(b) a peptide represented by an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added to any one of the amino acid sequences of SEQ ID NOs: 5 to 11, and the salt concentration dependence A peptide that exhibits self-assembly.

6. Gene encoding the peptide of (a) or (b)

(a) a peptide represented by the amino acid sequence of SEQ ID NO: 1 (b) a peptide represented by the amino acid sequence of SEQ ID NO: 1 wherein one or several amino acids have been deleted, substituted, and Z or added, and have a salt concentration-dependent self-assembly property. Indicating peptides

7. Gene encoding (a) or (b) peptide

(a) a peptide represented by the amino acid sequence of SEQ ID NO: 2

(b) A peptide represented by an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added to the amino acid sequence of SEQ ID NO: 2, and having a salt concentration-dependent self-assembly property. Indicating peptides.

8. Gene encoding the peptide of (a) or (b)

(a) a peptide represented by the amino acid sequence of SEQ ID NO: 3

(b) a peptide represented by the amino acid sequence of SEQ ID NO: 3 in which one or several amino acids have been deleted, substituted, and Z or added, and has a salt concentration-dependent self-assembly property. Peptide.

9. Gene encoding the peptide of (a) or (b)

(a) a peptide represented by the amino acid sequence of SEQ ID NO: 4

(b) a peptide represented by an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added to the amino acid sequence of SEQ ID NO: 4, and has a salt concentration-dependent self-assembly property. Peptide.

10. Gene encoding the peptide of (a) or (b)

(a) a peptide represented by any one of the amino acid sequences of SEQ ID NOS: 5 to 11;

(b) a peptide represented by an amino acid sequence in which one or several amino acids are deleted, substituted, and Z or added to any one of the amino acid sequences of SEQ ID NOs: 5 to 11, and the salt concentration dependence A peptide that exhibits self-assembly.

1 1. An expression vector incorporating the gene according to any one of claims 6 to 10.

1 2. The method for producing a peptide according to any one of claims 1 to 5, wherein the expression vector according to claim 11 is used.

1 3. A method of self-assembly of a peptide by adding the peptide according to any one of claims 1 to 5 to an aqueous solution containing a salt by adding the peptide to an aqueous solution containing the salt.

14. The method according to claim 13, wherein the salt concentration is 0.5 M or more.

15. A peptide assembly self-assembled by exposing the peptide according to any one of claims 1 to 5 under a high salt concentration.

16. The peptide aggregate according to claim 15, which is a film, a nanofiber, or a hydrogel.

17. One or several amino acids other than Cys in the (1) peptide of any one of SEQ ID NOs: 1 to 12 or (2) the amino acid sequence represented by SEQ ID NOs: 1 to 12 are deleted, substituted, and Or a peptide represented by an added amino acid sequence, wherein the peptide having salt concentration-dependent self-assembly properties is reacted with a salt under alkaline conditions to form a peptide aggregate having regular pores. How to form.

18. (1) one or several amino acids other than Cys in the amino acid sequence represented by (1) any one of SEQ ID NOS: 1 to 12 or (2) the amino acid sequence represented by SEQ ID NOs: 1 to 12 and / or A peptide represented by an added amino acid sequence, having regular pores formed by reacting a peptide exhibiting salt concentration-dependent self-assembly with a salt under alkaline conditions. Peptide aggregate.

19. The peptide assembly according to claim 18, which is a nanofiber or a nanomesh.