JP4303137B2 - Novel polypeptide and method for producing the same - Google Patents

Description

  The present invention relates to a novel polypeptide useful for promoting nucleation of apatites without the risk of pathogen infection and undesirable side effects, and a method for producing the same. More specifically, a highly safe biomaterial or biocompatible material, in particular, a novel polypeptide (including a polypeptide carrying an apatite) useful for the repair, repair and / or regeneration of a hard tissue, and a method for producing the same About.

  Collagen is a fibrous protein found in all multicellular animals and accounts for 25% of the total protein in mammals as the main component of skin and bone. A typical collagen molecule has a rope-like superhelical structure in which three collagen polypeptide chains are called a triple helical structure. Collagen contains a particularly large amount of proline (Pro) and glycine (Gly), and both amino acid residues are important for the formation of a stable triple helical structure.

  Examples of methods for using collagen as a biomaterial include, for example, a method in which porcine skin tissue is directly or lyophilized, and then transplanted to a damaged site of skin due to burns, a method in which cell components are removed by enzyme treatment, and the like. There is a method in which collagen solubilized by an acidic solution or enzyme treatment is reconstituted into a desired form. A general collagen preparation method and qualitative method are described in “Methods Enzymol., Vol. 82” (Non-patent Document 1), pp 33-64, 1982.

  Various proposals have been made on the use of collagen. For example, in Japanese Patent Application Laid-Open No. 08-027192 (Patent Document 1), in order to moisturize and smooth the skin, an animal tissue containing collagen is modified by esterification with alcohol, and then the modified collagen is added. A method for producing a collagen derivative to be extracted and a cosmetic base using the same have been proposed.

  Japanese Patent Application Laid-Open No. 07-097454 (Patent Document 2) discloses a method for subjecting soluble collagen to a triple after heat denaturation by crosslinking with an alkylenediimidate bivalent crosslinking reagent having imide ester groups at both ends of a methylene chain. A method for producing cross-linked collagen having a high helical structure regeneration rate and soluble in water is described.

  In Japanese Patent Application Laid-Open No. 08-053548 (Patent Document 3), collagen is reacted with a first synthetic hydrophilic polymer to form a collagen-synthetic polymer matrix, and the collagen-synthetic polymer matrix is further converted into a second synthetic hydrophilic polymer. Low immunogenicity by reacting with functional polymers, biologically active substances, glycosaminoglycans and their derivatives, chemical cross-linking agents, esterifying agents, amidating agents, acylating agents, amino acids, polypeptides, etc. A collagen-synthetic polymer matrix useful for the preparation of biocompatible implants for use in various medical applications has been described.

  Japanese Patent Application Laid-Open No. 07-278312 (Patent Document 4) describes a conjugate containing a hydrophilic synthetic polymer covalently bonded to chemically modified collagen having a substantially non-fiber form at pH 7. This document describes that the conjugate is particularly useful in ophthalmic devices and is optically transparent and biocompatible.

  In Japanese Patent Laid-Open No. 05-000158 (Patent Document 5), a collagen matrix is crushed, the crushed matrix is centrifuged in a high centrifugal field, a precipitate is homogenized to form a paste, the paste is cast, A method for producing a collagen film-like substance is described in which a molded paste is dried at a temperature of 37 ° C. or lower. It is also described that this collagen membrane material is biocompatible, non-inflammatory, and useful for tissue repair as an artificial implant.

  Japanese Patent Application Laid-Open No. 05-125100 (Patent Document 6) describes a high-purity soluble fish scale collagen and a method for producing the same by performing pepsin treatment as it is or after decalcifying fish scales.

  In Japanese Patent Laid-Open No. 06-228506 (Patent Document 7), a collagen solution is ejected from a nozzle in a 70 to 90% ethanol medium to form a filamentous or membranous material, dried, and then cut or pulverized A method for producing a granular or powdery soluble collagen dried product is described.

  In Japanese Patent Laid-Open No. 08-276003 (Patent Document 8), as a material for repairing living hard tissue such as bone, an unsintered hydroxyapatite single crystal is attached to at least a part of collagen fibers having a reduced antigenicity. The use is described.

  Japanese Patent Application Laid-Open No. 08-041425 (Patent Document 9) discloses a method of removing cells and tissue fragments from a collagen solution and performing an alkali treatment in order to remove prions in animal or human-derived collagen. Has been described.

  In addition, regarding the method of chemical synthesis of collagen analogues, p-nitrophenyl ester of Pro-Ser-Gly or p-nitrophenyl ester of Pro-Ala-Gly is dissolved in dimethylformamide, and triethylamine is added for 24 hours. It is reported that a soluble polyamide having a molecular weight of 16,000 to 21,000 can be obtained by placing ("J. Mol. Biol., Vol. 63" (Non-patent Document 2), pp. 85-99, 1972). . These soluble polyamides are estimated to have a triple helical structure from the circular dichroism spectrum, but there is no description regarding the properties of the obtained polymer.

  A 50-mer peptide containing the elastin-derived Val-Pro-Gly-Val-Gly sequence was dissolved in dimethyl sulfoxide, and 2 equivalents of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and 1 equivalent of 1 It has also been reported that after adding -hydroxybenzotriazole and 1.6 equivalents of N-methylmorpholine and allowing to stand for 14 days, a dialysis membrane having a molecular weight cutoff of 50,000 is dialyzed to obtain a polyamide ("" Int. J. Peptide Protein Res., Vol. 46 "(Non-Patent Document 3), pp. 453-463, 1995).

  JP-A-2003-321500 (Patent Document 10) discloses that a polypeptide composed of peptide units represented by the following formulas (1) to (3) can form a collagen tissue. Has been.

[-(OC- (CH ₂ ) _m -CO) _p- (Pro-Y-Gly) _n- ] _a (1)
[-(OC- (CH ₂ ) _m -CO) _q- (Z) _r- ] _b (2)
[-HN-R-NH-] _c (3)
(In the formula, m is an integer of 1 to 18, p and q are the same or different and 0 or 1, Y represents Pro or Hyp, n represents an integer of 1 to 20. Z represents 1 to 10 amino acids. Represents a peptide chain consisting of residues, r represents an integer of 1 to 20, R represents a linear or branched alkylene group, and the ratio of a to b is a / b = 100/0 to 30 /. 70 (molar ratio), c = a when p = 1 and q = 0, c = b when p = 0 and q = 1, and c = p when p = 1 and q = 1. a + b, c = 0 when p = 0 and q = 0.)
As described in Patent Document 9, the causative substance of sheep tremor and bovine spongiform encephalopathy is an infectious protein called prion, and this infectious protein is an infectious disease of human Creutzfelder-Jakob disease. It is said to be one of the causes. It is pointed out that prion is a protein, hardly inactivated by ordinary sterilization and sterilization methods, and infects beyond species ("Nature Review, Vol. 2" (Non-patent Document 4), pp. 118-126, 2001).

  In general, collagens derived from cattle and pigs are often used as raw materials in medical devices, pharmaceuticals, and cosmetics. Therefore, there is always a risk of infection (or transmission) of pathogens (or pathogenic factors) such as prions that cannot be removed by ordinary sterilization and sterilization methods.

  In addition, since natural collagen contains various cell adhesion sites, cell selectivity according to the application cannot be exhibited. For example, when collagen is used as a nerve axon guidance material, the migration and proliferation rate of surrounding fibroblasts is greater than the rate of axon extension, and the axon cannot be elongated due to scar organization. For this reason, it is necessary to take measures such as covering the periphery of collagen with a material that prevents migration of fibroblasts.

  On the other hand, in a living body, certain ceramics (for example, Bioglass (registered trademark) as a bioactive glass, crystallized glass A-W (Cerabone (registered trademark) A-W), etc.) may bind to bone. Are known. This bonding between the ceramic and the bone is caused by the formation of a hydroxyapatite layer on the surface of the ceramic in vivo (or in an aqueous solution having an ionic concentration close to that of human body fluid). First, the silicate ions and silanol groups formed on the ceramic surface react with calcium and phosphate ions in the living body and aqueous solution to form hydroxyapatite nuclei. It is thought to grow by taking in supersaturated calcium and phosphate ions in vivo or in aqueous solution.

  In JP-A-5-103829 (Patent Document 11), a liquid silica hydrosol or gel is coated on a substrate of various shapes such as plate, rod, fiber and granule, and dried. Then, after the silica gel is bonded to the substrate by heat treatment, it is immersed in an aqueous solution (pseudo body fluid) containing calcium and phosphate ions in an amount that is supersaturated with respect to hydroxyapatite. Bioactive layer coating methods for coating layers have been proposed. This document also describes that the apatite coating material can be used for artificial bones, bioimplantable materials, bioimplantable medical devices, instruments, and the like. However, such inorganic biomaterials have insufficient biocompatibility such as cell adhesion.

In addition, organic-inorganic composite materials as biomaterials are also being studied. For example, in Japanese Patent Application Laid-Open No. 2003-190271 (Patent Document 12), hydroxyapatite and collagen having an average fiber length of 60 μm or more (collagen or collagen-like protein obtained from mammals, birds, fish, etc., genetically modified collagen, etc.) An organic-inorganic composite biomaterial composed of a composite containing Further, in this document, the composite material was obtained by maintaining calcium ions and phosphate ions in a reaction vessel at a specific concentration, for example, by controlling the starting material concentration and the liquid feeding speed. It is also described that the composite can be produced by pressing. “Chem. Mater. 15”, pp3221-3226, 2003 (Non-patent Document 5) describes rat tail tendon solubilized with acid in the presence of 0.1 M CaCl ₂ and 0.1 M NaH ₂ PO _4. A method is described in which collagen and hydroxyapatite are combined by neutralizing the derived collagen.

  However, even in such a complex, there is a risk of infection (or transmission) of a pathogen (or pathogenic factor) if natural collagen is used as the collagen.

JP-A-2003-154001 (Patent Document 13) discloses a method for producing a composite by bringing an aqueous solution containing calcium ions and phosphate ions into contact with a base having sericin and depositing apatites on the base. It is disclosed.
Japanese Patent Laid-Open No. 08-027192 Japanese Patent Application Laid-Open No. 07-097454 Japanese Patent Laid-Open No. 08-053548 JP 07-27831 A Japanese Patent Laid-Open No. 05-000158 JP 05-125100 A Japanese Patent Laid-Open No. 06-228506 Japanese Patent Laid-Open No. 08-276003 Japanese Patent Laid-Open No. 08-041425 JP 2003-321500 A (Claim 1, paragraph number [0043]) JP-A-5-103829 (Claim 1) JP 2003-190271 A (claims 1, 4 and 6, paragraph number [0014]) JP2003-154001A (Claim 1) "Methods Enzymol., Vol.82", pp33-64, 1982 "J. Mol. Biol., Vol. 63", pp.85-99, 1972 "Int. J. Peptide Protein Res., Vol.46", pp.453-463, 1995 "Nature Review, Vol.2", pp.118-126, 2001 "Chem. Mater. 15", pp3221-3226, 2003

  Accordingly, an object of the present invention is to provide a novel polypeptide useful as a highly safe biomaterial or biocompatible material without the risk of causing infection with a pathogen or transmission of a pathogenic factor or an undesirable side effect, and a method for producing the same. Is to provide.

  Another object of the present invention is to provide a polypeptide useful for supplementing, repairing and / or regenerating living hard tissue.

  As a result of intensive studies to solve the above problems, the inventor has obtained a collagen-like polypeptide (fibrous aggregate) obtained by condensing a peptide component containing a specific amino acid sequence in a living body or a living body-similar environment. The inventors have found that hydroxyapatite is deposited on the polypeptide, and completed the present invention.

  That is, the novel polypeptide of the present invention comprises a peptide unit (1) having an amino acid sequence represented by the following formula (1) and a peptide unit having an amino acid sequence represented by the following formula (2). Yes.

-Pro-X-Gly- (1)
-YZ-Gly- (2)
(Wherein X and Z are the same or different and each represents Pro or Hyp, and Y represents an amino acid residue having a carboxyl group (for example, an α-amino acid residue))
In the formula (2), Y may be Asp or Glu which may have a carboxyl group at the γ position.

  The ratio (molar ratio) between the peptide unit (1) and the peptide unit (2) may be about (1) / (2) = 99/1 to 1/99.

The polypeptide of the present invention exhibits a positive cotton effect at a wavelength of 220 to 230 nm and a negative cotton effect at a wavelength of 195 to 205 nm in a circular dichroism spectrum. This indicates that at least a part (part or all) of the polypeptide forms a triple helical structure. The polypeptide may exhibit a peak in the molecular weight range of 5 × 10 ^{3 to} 500 × 10 ⁴ . The polypeptide can form a collagen tissue (collagen-like fibrous aggregate). The polypeptide may carry apatites (such as hydroxyapatite).

  In the present invention, the polypeptide is obtained by condensing an amino acid component or peptide fragment component containing at least an amino acid or peptide fragment corresponding to the formula (1) and an amino acid or peptide fragment corresponding to the formula (2). Obtainable. Examples of the polypeptide include (a) a method of condensing a peptide component containing at least a peptide having both amino acid sequences represented by the formulas (1) and (2), and (b) a formula represented by the formula (1). It may be produced by a method of condensing a peptide component containing at least a peptide having the amino acid sequence represented by formula (2) and a peptide component containing the peptide having the amino acid sequence represented by formula (2).

The polypeptide (especially its fibrous aggregate) can form a complex (hybrid) of the polypeptide and apatite by depositing apatites (such as hydroxyapatite) in a body fluid-like environment, for example. . In the present invention, a polypeptide carrying the apatite can be produced by bringing the polypeptide into contact with an aqueous solution containing calcium ions and phosphate ions to deposit apatites on the polypeptide. The present invention also includes a polypeptide carrying an apatite obtained by such a production method.
The present invention also includes cosmetic materials and food additives containing the polypeptide.

  Since the novel polypeptide of the present invention has a specific amino acid sequence, it has a risk of causing infection of a pathogen or transmission of a virulence factor or an undesirable side effect despite having excellent cell affinity and biocompatibility. There is no safety. In addition, the polypeptide (particularly a fibrous aggregate of the polypeptide) is used to supplement or repair a biological hard tissue by depositing or depositing apatites in a living body or in a living body-like environment (such as a body fluid-like environment). And / or complexes of polypeptides useful for regeneration and apatites can be formed. Such polypeptides and complexes are suitable for biomaterials or biocompatible materials.

  In the present invention, various amino acid residues are described by the following abbreviations.

Ala: L-alanine residue
Arg: L-arginine residue
Asn: L-asparagine residue
Asp: L-aspartic acid residue
Cys: L-cysteine residue
Gln: L-glutamine residue
Glu: L-glutamic acid residue
Gly: Glycine residue
His: L-histidine residue
Hyp: L-hydroxyproline residue
Ile: L-isoleucine residue
Leu: L-leucine residue
Lys: L-lysine residue
Met: L-methionine residue
Phe: L-phenylalanine residue
Pro: L-proline residue
Sar: Sarcosine residue
Ser: L-serine residue
Thr: L-threonine residue
Trp: L-tryptophan residue
Tyr: L-tyrosine residue
Val: L-valine residue In the present specification, according to a conventional method, the amino acid sequence of the peptide chain is determined by positioning the N-terminal amino acid residue on the left side and the C-terminal amino acid residue on the right side. Describe.

[New polypeptide]
The novel polypeptide of the present invention needs to contain a peptide unit having an amino acid sequence represented by -Pro-X-Gly- (1). Since the sequence represented by -Pro-X-Gly- contributes to the stability of the triple helix structure, the stability of the triple helix structure decreases when the ratio of this sequence is small. Furthermore, this unit forms a repeating structure (oligo or polypeptide unit structure) represented by-(Pro-X-Gly) _n -in the polypeptide from the viewpoint of the stability of the triple helical structure. Also good. The repeating number n of this arrangement is, for example, about 1 to 5000, preferably about 2 to 3000. X may be either Pro or Hyp, but is more preferably Hyp in view of the stability of the triple helical structure. Hyp is usually a 4Hyp (eg, trans-4-hydroxy-L-proline) residue.

  Further, the polypeptide of the present invention needs to contain a peptide unit having an amino acid sequence represented by -Y-X-Gly- (2). By having such a peptide unit (2), apatites (such as hydroxyapatite) can be efficiently precipitated or supported on the polypeptide.

  In the formula (2), Y may be an amino acid residue having a carboxyl group (for example, an α-amino acid residue having a carboxyl group). Examples of such amino acid residues include Glu and Asp. Glu may have a carboxyl group at the γ position. Glu having a carboxyl group at the γ-position is represented by a γ-carboxyglutamic acid residue Gla. In the formula (2), Z may be either Pro or Hyp.

  Furthermore, the polypeptide of the present invention may contain other amino acid residues and peptide residues (units) as long as the physical and biological properties of the resulting polypeptide are not impaired. In order for the polypeptides of the present invention to exhibit useful physical and biological properties, for example, Gly, Sar, Ser, Glu, Asp, Lys, His, Ala, Val, Leu, Arg, Pro, Tyr, At least one amino acid residue or peptide residue selected from Ile, Thr, and Cys, in particular, at least one amino acid residue selected from Gly, Sar, Ser, Glu, Asp, Lys, Arg, Pro, Val Or it often has a peptide residue. Specifically, for example, Gly, Sar, Ser, Glu, Asp, Lys, Arg-Gly-Asp, Tyr-Ile-Gly-Ser-Arg, Ile-Lys-Val-Ala-Val, Val-Pro-Gly -Val-Gly, Asp-Gly-Glu-Ala, Gly-Ile-Ala-Gly, His-Ala-Val, Glu-Arg-Leu-Glu, Lys-Asp-Pro-Lys-Arg-Leu, Arg-Ser It preferably contains an amino acid residue or peptide residue represented by -Arg-Lys.

  The polypeptide of the present invention may be a physiologically or pharmacologically acceptable salt. For example, inorganic acid (hydrochloric acid, sulfuric acid, phosphoric acid, etc.), organic acid (acetic acid, trifluoroacetic acid, lactic acid, tartaric acid) , Maleic acid, fumaric acid, oxalic acid, malic acid, citric acid, oleic acid, palmitic acid, etc., metals (alkali metals such as sodium and potassium, alkaline earth metals such as calcium, aluminum, etc.), organic bases (trimethylamine) , Triethylamine, t-butylamine, benzylamine, diethanolamine, dicyclohexylamine, arginine and the like. These salt-forming compounds can be used alone or in combination of two or more. These salts can be obtained by ordinary salt formation reactions.

  In the polypeptide of the present invention, the ratio (molar ratio) between the peptide unit (1) and the peptide unit (2) is (1) / (2) = 99/1 to 1/99, preferably 98/2. ˜2 / 98, more preferably about 95/5 to 5/95.

  The ratio (molar ratio) between the total amount of the peptide unit (1) and the peptide unit (2) and other peptide units is the former / the latter = 100/0 to 50/50, preferably 100/0 to 60. / 40, more preferably about 100/0 to 70/30.

Such a polypeptide forms a chain polypeptide without forming a 6-membered ring by cyclization, and a solvent (water, sulfoxides such as dimethyl sulfoxide, dimethylformamide, dimethylacetamide, N-methyl). It is soluble or partially soluble in a hydrophilic solvent such as pyrrolidone or a mixed solvent thereof. In the aqueous gel permeation chromatography (GPC), the polypeptide of the present invention has a molecular weight of, for example, 5 × 10 ^{3 to} 500 × 10 ⁴ , preferably 1 × 10 ^{4 to} 300 × 10 ⁴ , preferably in terms of globular protein. Shows a peak in the range of about 3 × 10 ⁴ to 200 × 10 ⁴ , more preferably about 5 × 10 ^{4 to} 100 × 10 ⁴ .

  Furthermore, the polypeptide of the present invention exhibits a positive cotton effect at a wavelength of 220 to 230 nm and a negative cotton effect at a wavelength of 195 to 205 nm in a circular dichroism spectrum. Therefore, at least a part (that is, part or all) of the polypeptide can form a triple helical structure, forming a collagen-like polypeptide. The cotton effect refers to a phenomenon that occurs because the optical rotation material has different absorption coefficients for left and right circularly polarized light at a specific wavelength.

  The polypeptide of the present invention can form a collagen-like fibrous aggregate structure. Polypeptide chains forming the above triple helical structure self-assemble to form a fibrillar structure of several nm to several tens of nm, and these fibrils are arranged to form a fiber structure of several μm to several tens of μm. Can be formed. These can be observed with a transmission electron microscope, a scanning electron microscope, or an atomic force microscope.

  The polypeptide can precipitate apatites (such as hydroxyapatite) on the polypeptide (particularly a fibrous aggregate) in a body fluid or a body fluid-like environment (or a body-like environment).

  The polypeptide may carry apatites. Such a polypeptide (a complex of a polypeptide and an apatite (hereinafter sometimes simply referred to as a complex)) is also included in the polypeptide of the present invention.

A representative compound of the apatites is hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ), but the apatites are calcium phosphate compounds such as calcium hydrogen phosphate (CaHPO ₄ ), calcium phosphate ( Ca ₃ (PO ₄ ) ₂ ) and the like are also included.

  The amount of apatite supported can be selected from a wide range, for example, from about 0 to 5000 parts by weight, preferably 0.1 to 3000 parts by weight, with respect to 100 parts by weight of the polypeptide, depending on the application. More preferably, it may be about 1 to 1000 parts by weight.

  The polypeptide in the polypeptide or complex may be cross-linked with a cross-linking agent as necessary. Examples of the crosslinking agent include dialdehydes such as glyoxal, glutaraldehyde, and succinaldehyde, and physiologically acceptable crosslinking agents such as dextran dialdehyde and aldehyde starch. The proportion of the cross-linking agent may be about 1 to 20 parts by weight, preferably about 1 to 10 parts by weight with respect to 100 parts by weight of the polypeptide.

  Since the polypeptide (including the complex) of the present invention is chemically synthesized from a pure reagent, it can be used for pathogens and pathogenic factors [eg, proteins converted to pathogenicity (eg, abnormal prions, etc.)]. There is no risk of infection or transmission. Therefore, the polypeptide of the present invention is highly safe. It is also excellent in cell affinity and biocompatibility. Therefore, it is useful as a biomaterial or a biocompatible material, for example, artificial collagen.

  The polypeptide can be used effectively in, for example, repairing, repairing, and regenerating defects in hard tissues such as bone and cartilage. In such a use, the polypeptide may be used as it is, for example, in the form of a fibrous aggregate, or in the environment similar to a body fluid, the polypeptide (fibrous aggregate) and apatite may be used. The complex may be formed in advance and used in the state of this complex.

  The polypeptide (including complex) of the present invention can be applied to a tissue (eg, bone, cartilage, etc.) of a subject (subject). Examples of the subject include humans and non-human animals (for example, non-human animals such as monkeys, sheep, cows, horses, dogs, cats, rabbits, rats, mice).

  In the present invention, the usage form of the polypeptide (including the complex) is not particularly limited, and for example, a liquid (solution or suspension), powder, or one-dimensional base material (fibrous or thread-like) Base material, linear base material, rod-like base material), two-dimensional base material (film (or sheet) or plate-like base material)), three-dimensional base material (tubular base material, etc.) It may be. Further, the solid polypeptide (including the complex) may be non-porous or porous (for example, a two-dimensional porous material such as a granular porous material, a nonwoven fabric or a woven fabric, a cylindrical shape, etc. Or a three-dimensional porous body).

  The said polypeptide (a complex is also included) can be made into a desired form by shape | molding by a conventional method according to a use. For example, a solution or suspension of a polypeptide (or complex) is cast on a peelable substrate (for example, a fluororesin (polytetrafluoroethylene) sheet), dried, and peeled from the substrate. By doing so, a sheet or film of a polypeptide or a complex can be obtained. In addition, a fibrous material can be obtained by extruding a solution or suspension of a polypeptide or complex from a nozzle in a poor solvent for the polypeptide or complex (or a solution containing a high concentration of salt, etc.). . Furthermore, a gel-like substance can be obtained by allowing an aqueous solution or suspension of a polypeptide or a complex to stand, or if necessary, adding a polyvalent crosslinking reagent (such as glutaraldehyde) and allowing to stand. Furthermore, a sponge-like porous body can be obtained by freeze-drying the generated gel. Furthermore, a porous body can also be obtained by foaming (foaming) an aqueous solution or suspension of the polypeptide or complex by stirring or the like and drying.

  Furthermore, the polypeptide (including the complex) may be used as a coating agent. For example, the surface of the substrate may be coated by applying or spreading a solution or suspension of the polypeptide (or complex) on the surface of the substrate and then drying. Examples of the substrate include molded bodies formed of various materials such as metals, ceramics, and polymers (synthetic or natural polymers). In addition, base materials, such as a polymer molded object, may have biodegradability or biodegradability.

  The form of the formed body is not particularly limited, and is granular, one-dimensional shaped substrate (fibrous or thread-like substrate, linear substrate, rod-shaped substrate, etc.), two-dimensional shaped substrate (film) (Or a sheet) or a plate-like substrate), a three-dimensional substrate (such as a tube-like substrate), and the like. Further, the substrate may be a non-porous body (for example, a granular porous body, a cellulose fiber paper, a two-dimensional porous body such as a nonwoven fabric or a woven fabric, a three-dimensional body such as a cylindrical shape). Porous body). In such a porous substrate, a polypeptide or a complex solution or suspension may be impregnated to hold the polypeptide or its complex with apatite. If necessary, the substrate may be surface-treated with a surface treatment agent (for example, a physiologically acceptable surface treatment agent).

  The polypeptide (including the complex) may be sterilized or sterilized as necessary. In particular, in medical applications and the like, usually, a polypeptide or a complex is often sterilized or sterilized. As the sterilization and sterilization methods, conventional methods such as wet heat steam sterilization, gamma ray sterilization, ethylene oxide gas sterilization, chemical sterilization, and ultraviolet sterilization can be used. Among these methods, gamma ray sterilization and ethylene oxide gas sterilization are preferable in that they have a small effect on sterilization efficiency and materials.

(Method for producing polypeptide)
The novel polypeptide of the present invention can be obtained by a conventional method in which amino acids and peptide fragments (or segments) are subjected to a condensation reaction. Finally, peptide unit (1) and peptide unit (2) are contained in the polypeptide. Is not particularly limited as long as it is contained in, for example, may be obtained by using a condensation reaction of constituent amino acids or a condensation reaction of a peptide fragment and an amino acid, but may be obtained in advance by the formula (1) and / or (2) It is preferable to obtain by the method of preparing the peptide which has an amino acid sequence represented by these, and condensing the peptide component containing this peptide.

  In the method of condensing a peptide component prepared in advance, the peptide chain of the peptide component can be synthesized according to an ordinary peptide synthesis method. The peptide can be prepared, for example, by a solid phase synthesis method or a liquid phase synthesis method, but the solid phase synthesis method is simple in operation [for example, “Sequence Chemistry Laboratory Lecture 2 Protein Chemistry (Lower)” edited by the Japanese Biochemical Society) (See May 20, 1987, issued by Tokyo Chemical Co., Ltd.), pages 641-694]. For peptide synthesis, conventional methods such as coupling methods using condensing agents, active ester methods (phenyl esters such as p-nitrophenyl ester (ONp) and pentafluorophenyl ester (Opfp), N-hydroxysuccinimide ester ( N-hydroxydicarboxylic acid imide ester such as ONSu), 1-hydroxybenzotriazole ester (Obt), etc.), mixed acid anhydride method, azide method and the like can be used. In a preferred method, at least a condensing agent (preferably a condensing agent described later, particularly a combination of a condensing agent and a condensing aid described later) is often used.

  Furthermore, in peptide synthesis, depending on the type of amino acid or peptide segment, depending on the protecting group of amino group, carboxyl group, and other functional groups (guanidino group, imidazolyl group, mercapto group, hydroxyl group, ω-carboxyl group, etc.) Protection and elimination / removal of the protective group by catalytic reduction or strong acid treatment (anhydrous hydrogen fluoride, trifluoromethanesulfonic acid, trifluoroacetic acid, etc.) are repeated. For example, amino-protecting groups include benzyloxycarbonyl group (Z), p-methoxybenzyloxycarbonyl group (Z (OMe)), 9-fluorenylmethoxycarbonyl group (Fmoc), t-butoxycarbonyl group ( Boc), 3-nitro-2-pyridinesulfenyl group (Npys) and the like, and the protective group for carboxyl group is benzyloxy group (OBzl), phenacyloxy group (OPac), t-butoxy group (OBu ), Methoxy group (OMe), ethoxy group (OEt), and the like. An automatic synthesizer may be used for peptide synthesis.

  More specifically, the peptide chain can be prepared by a solid phase synthesis method by a conventional method. Examples of the solid phase resin (or carrier) include polymers insoluble in the reaction solvent, such as styrene-divinylbenzene copolymer, such as chloromethyl resin, hydroxymethyl resin, hydroxymethylphenylacetamidomethyl resin, 4-methylbenzhydride. Luamine resin can be used.

  In the solid-phase synthesis method, usually (i) the polymer (resin) has a free α-COOH group and a functional group (at least at the C-terminal to N-terminal direction of the target peptide). (Ii) an α-amino group protecting group that forms a peptide bond among the bound amino acids or peptide fragments; (Iii) a step of extending the peptide chain to form a peptide chain corresponding to the target peptide by sequentially repeating the above binding operation and the removing operation; and (iv) a peptide chain that is a polymer (resin ) And removing the protecting group from the protected functional group to produce the desired peptide, and purify the produced peptide to produce the peptide. In the operation (i) for binding the amino acid or peptide fragment, an amino acid (for example, Fmoc-) corresponding to the C-terminus of the peptide chain and having a free α-COOH group and at least the N-terminus protected with a protecting group. Amino acids, Boc-amino acids, etc.) are used. The elimination of the peptide chain from the polymer and the removal of the protecting group are preferably performed simultaneously using trifluoroacetic acid from the viewpoint of suppressing side reactions. Further, the produced peptide can be purified using a separation and purification means such as reverse phase liquid chromatography or gel permeation chromatography.

  A method for condensing peptide components containing peptides synthesized in this manner includes (a) a method for condensing peptide components containing at least peptides having both amino acid sequences represented by formulas (1) and (2). And (b) a method of condensing a peptide component comprising at least a peptide having the amino acid sequence represented by formula (1) and a peptide having the amino acid sequence represented by formula (2).

  In the former method (a), a peptide having both amino acid sequences represented by formulas (1) and (2) (that is, a peptide unit having an amino acid sequence represented by formula (1) and formula (2) Peptides containing both units with the peptide unit having the amino acid sequence represented can be used alone or in combination of two or more. In this method, as the peptide component, in addition to the peptide containing both of the above units, other peptides may be used depending on the target polypeptide. Examples of other peptides include the above-mentioned other amino acid residues and peptides containing peptide residues. These other peptides can also be used alone or in combination of two or more. In this method, the ratio between the unit (1) and the unit (2) can be easily adjusted by co-condensing the peptide having the amino acid sequence represented by the formula (1) or (2). .

  Also in the latter method (b), the peptide (oligo or polypeptide unit) (1) having the amino acid sequence represented by the formula (1) (1) and the peptide (2) having the amino acid sequence represented by the formula (2) are: These can be used alone or in combination of two or more. Also in this method, as the peptide component, in addition to these peptides (1) and (2), other peptides such as the other amino acid residues and peptide residues described above according to the target polypeptide Peptides containing etc. may be used. These other peptides can also be used alone or in combination of two or more.

  The condensation reaction of these peptides is usually performed in a solvent. The solvent may be any solvent as long as it can dissolve or suspend (partially or completely dissolve) the peptide component and the compound, and water and / or an organic solvent can be usually used. Examples of the solvent include water, amides (dimethylformamide, dimethylacetamide, hexamethylphosphoramide, etc.), sulfoxides (dimethylsulfoxide, etc.), nitrogen-containing cyclic compounds (N-methylpyrrolidone, pyridine, etc.), nitriles ( Examples include acetonitrile (such as acetonitrile), ethers (such as dioxane and tetrahydrofuran), alcohols (such as methyl alcohol, ethyl alcohol, and propyl alcohol), and mixed solvents thereof. Of these solvents, water, dimethylformamide, and dimethyl sulfoxide are frequently used.

  The reaction of these peptides can usually be carried out in the presence of at least a dehydrating agent (dehydrating condensing agent) or a condensing agent. When reacted in the presence of a dehydrating condensing agent and a condensing aid, dimerization or cyclization occurs. A polypeptide can be produced smoothly while suppressing the above.

  The dehydrating condensing agent is not particularly limited as long as dehydrating condensation can be efficiently performed in the solvent. For example, a carbodiimide condensing agent [diisopropylcarbodiimide (DIPC), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide ( EDC = WSCI), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (WSCI · HCl), dicyclohexylcarbodiimide (DCC), etc.], fluorophosphate condensing agent [O- (7-azabenzotriazole -1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate, O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyluronium hexafluorophosphate, benzo Triazol-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate Benzotriazol-1-yl - tris (dimethylamino) phosphonium hexafluorophosphate product salt (BOP)], such as diphenylphosphoryl azide (DPPA) can be exemplified. These dehydration condensing agents can be used alone or in combination of two or more. A preferred dehydrating condensing agent is a carbodiimide condensing agent [for example, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride].

  The condensation aid is not particularly limited as long as it promotes the reaction of the condensation agent. For example, N-hydroxypolycarboxylic imides [for example, N-hydroxysuccinimide (HONSu), N-hydroxy-5-norbornene N-hydroxydicarboxylic imides such as -2,3-dicarboxylic acid imide (HONB)], N-hydroxytriazoles [for example, N-hydroxybenzotriazoles such as 1-hydroxybenzotriazole (HOBt)], 3- Examples thereof include triazines such as hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HOObt), and 2-hydroxyimino-2-cyanoacetic acid ethyl ester. These condensation aids can also be used alone or in combination. Preferred condensation aids are N-hydroxydicarboxylic imides [such as HONSu], N-hydroxybenzotriazole or N-hydroxybenzotriazines [such as HOBt].

  The dehydration condensation agent and the condensation aid can be used in appropriate combination. Examples of the combination of the dehydrating condensing agent and the condensing aid include DCC-HONSu (HOBt or HOObt), WSCI-HONSu (HOBt or HOObt), and the like.

  The amount of the dehydrating condensing agent used is usually 0.7 to 5 mol, preferably 0.8 to 2.5 mol, more preferably, when a non-aqueous solvent not containing water is used per 1 mol of the total amount of the peptide. Is about 0.9-2.3 mol (for example, 1-2 mol). In a solvent containing water (aqueous solvent), since the dehydration condensing agent is deactivated by water, the amount of the dehydrating condensing agent used is generally 2 to 500 mol (for example, 2 to 50 mol), preferably 5 to 250 mol (for example, 5 to 25 mol), more preferably about 10 to 125 mol (for example, 10 to 20 mol). The amount of the condensation aid used is, for example, 0.5 to 5 moles, preferably 0.7 to 2 moles, and more preferably 0.8 to 2 moles per mole of the peptide regardless of the type of solvent. About 1.5 moles.

  In the condensation reaction of the present invention, the pH of the reaction system may be adjusted, or a base that does not participate in the reaction may be added. The pH can be adjusted usually using an inorganic base (sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogen carbonate, etc.), an organic base, an inorganic acid (hydrochloric acid, etc.) or an organic acid. The pH of the solution is adjusted to near neutral (pH = about 6 to 8). Examples of the base not involved in the reaction include tertiary amines such as trialkylamines such as trimethylamine, triethylamine and diisopropylethylamine, and heterocyclic tertiary amines such as N-methylmorpholine and pyridine. . The amount of such base used can usually be selected from a range of about 1 to 2 times the total number of moles of the peptide.

  The formation of a triple helical structure by the polypeptide of the present invention can usually be verified by measuring a circular dichroism spectrum of a solution of the polypeptide. In particular, in the circular dichroism spectrum, natural collagen and peptide chains forming a triple helical structure characteristically show a positive cotton effect at wavelengths of 220 nm to 230 nm and a negative cotton effect at wavelengths of 195 nm to 205 nm. (J. Mol. Biol., Vol. 63 pp. 85-99, 1972).

  In the present invention, the apatite is supported (deposited or deposited) on the polypeptide (particularly a fibrous aggregate) by bringing the polypeptide into contact with an aqueous solution (or pseudo body fluid) containing calcium ions and phosphate ions. And a complex of the polypeptide and apatites may be formed.

  In such a method, the calcium ions can be introduced into the aqueous solution by using various calcium compounds. Examples of calcium compounds include calcium halide (such as calcium chloride), calcium hydroxide, calcium oxide, inorganic acid salts (such as calcium nitrate, calcium sulfate, calcium carbonate, and calcium hydrogen carbonate), and organic acid salts (such as calcium acetate). Etc. can be exemplified. These calcium compounds can be used alone or in combination of two or more.

  Phosphate ions can also be introduced into the aqueous solution by using various phosphate components. Examples of such a phosphoric acid component include orthophosphoric acid or a salt thereof (an alkali metal phosphate such as sodium phosphate or potassium phosphate; an alkali metal hydrogen phosphate such as sodium hydrogen phosphate or potassium hydrogen phosphate; Examples thereof include alkaline earth metal salts of hydrogen phosphate such as calcium hydrogen phosphate (such as calcium dihydrogen phosphate; ammonium salts such as ammonium phosphate, ammonium hydrogen phosphate, and sodium ammonium hydrogen phosphate). These phosphoric acid components can also be used alone or in combination of two or more.

  The concentration of calcium ions and phosphate ions in the aqueous solution is not particularly limited as long as the efficiency of supporting (precipitation or deposition) of apatites is not lowered, but in the present invention, the concentration of calcium ions and phosphate ions is low in an aqueous solution. However, the apatites can be efficiently supported on the polypeptide. The aqueous solution preferably contains a supersaturated amount of calcium ions and phosphate ions with respect to calcium phosphate (that is, contains calcium ions and phosphate ions at a concentration exceeding the solubility of calcium phosphate). In addition, although it is preferable that calcium phosphate does not precipitate in aqueous solution, as long as the aqueous phase is supersaturated, calcium phosphate may precipitate.

  For example, at a temperature of 36.5 ° C., the calcium ion concentration in the aqueous solution can be selected from the range of about 2 to 50 mM (mmol / L), usually 2.5 mM or more (for example, about 2.5 to 30 mM), Preferably, it may be about 2.5 to 20 mM, more preferably about 3 to 10 mM. In addition, at a temperature of 36.5 ° C., the concentration of phosphate ions in the aqueous solution can be selected from a range of about 0.5 to 50 mM (mmol / L), and usually 1 mM or more (for example, about 1 to 30 mM), Preferably it may be about 1 to 20 mM, more preferably about 1.2 to 10 mM.

  The aqueous solution may contain other ion species such as metal ions [alkali metal ions (sodium, potassium ions, etc.), alkaline earth metal ions (magnesium ions, etc.), transition metal ions (titanium ions, zirconium ions, cobalt ions, etc.). ), Silicon ions, etc.], halogen ions (bromine anions, chlorine anions, fluorine anions, etc.), inorganic acid ions (for example, carbonate ions, bicarbonate ions, sulfate ions, etc.) may be included. These ionic species can be selected singly or in combination of two or more in accordance with the composition and crystal structure of the calcium phosphate compound.

  The aqueous solution containing calcium ions and phosphate ions may be adjusted with a buffer. As the buffer, Tris (tris (hydroxymethyl) aminomethane) buffer, phosphate buffer, borate buffer, carbonate buffer, citrate buffer, acetate buffer, and the like can be used. The pH of the aqueous solution is usually about 6-8.

Exemplary aqueous solutions include solutions having the following composition:
^{(1) Na + 142mM, K} + 5mM, Ca 2+ 2.5mM, Mg 2+ 1.5mM, C1 - 148.8mM, HCO 3 - 4.2mM, HPO 4 2- 1mM, SO 4 2- 0. An aqueous solution containing 5 mM, adjusted to pH 7.4 with Tris buffer,
(2) Na ⁺ 213 mM, K ⁺ 7.5 mM, Ca ²⁺ 3.8 mM, Mg ²⁺ 2.3 mM, Cl ⁻ 223.3 mM, HCO ₃ ⁻ 6.3 mM, HPO ₄ ²⁻ 1.5 mM, SO ₄ ^2- An aqueous solution containing 0.75 mM and adjusted to pH 7.4 with Tris buffer.

  The method of bringing the polypeptide of the present invention into contact with the aqueous solution may be any method that allows the polypeptide to be coated with apatites (calcium phosphate compounds), and may be spraying, impregnation, coating, etc. The method of contacting is simple.

  The contact temperature (for example, immersion temperature) between the polypeptide and the aqueous solution can be appropriately selected from the range of 10 to 100 ° C. depending on the apatite (calcium phosphate compound) to be coated, and is usually 10 to 60 ° C., preferably 20 It is about -50 degreeC, More preferably, it is about 25-45 degreeC (for example, 30-39 degreeC). The contact time (for example, immersion time) can be selected according to the type of apatite and the target deposition amount, and is usually within 10 days (particularly within 7 days). In addition, when contacting with polypeptide in aqueous solution, although it stirs normally normally, it may stand still and apatite may be deposited.

  Polypeptides and composites of the present invention include artificial bones, artificial tooth roots, bone implants such as bone repair agents, bone fillers, tissue engineering carriers or supports for hard tissues, and regenerative medicine carriers or supports. It can be used for medical materials such as the body, pharmaceutical preparation materials (eg, bases, additives, etc.), cosmetic materials (eg, bases, additives, etc.), food additives, coating agents, and the like.

  Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.

Example 1
The peptide chain represented by the formula: H- (Pro-Hyp-Gly) ₄ -Glu-Hyp-Gly- (Pro-Hyp-Gly) ₅ -OH (SEQ ID NO: 1) is immobilized using an automatic peptide synthesizer. It was synthesized by the phase synthesis method. That is, a styrene-divinylbenzene copolymer containing 4- (Nα-9- (fluorenylmethoxycarbonyl) -glycine) -oxymethyl-phenoxy-methyl group at a ratio of 0.65 mmol / g (resin) [styrene and divinyl Using 0.1 mmol of granular resin (HMP glycine, manufactured by Applied Biosystems, USA) formed in a molar ratio with benzene = 99: 1], the corresponding peptides are sequentially handled from the carboxyl terminus to the amino terminus. Amino acids were conjugated. In the coupling reaction, Nα-9- (fluorenylmethoxycarbonyl) -L-proline [Fmoc proline], Nα-9- (fluorenylmethoxycarbonyl) -glycine [Fmoc] manufactured by Applied Biosystems, Inc. Glycine], Nα-9- (fluorenylmethoxycarbonyl) -γ-t-butyloxy-L-glutamic acid [Fmoc glutamic acid], Nα-9- (fluorenylmethoxycarbonyl) -Ot-butyl manufactured by Bacchem 1 mmol each of -L-hydroxyproline [Fmoc hydroxyproline] was used for each coupling step.

  The obtained peptide resin was treated with 10 mL of trifluoroacetic acid containing 5% water for 3 hours. The resulting solution was added to diethyl ether, and the resulting precipitate was further washed several times with diethyl ether to deprotect the peptide and desorb from the resin. The crude product was purified with a PD10 column (manufactured by Amersham Biosciences) to obtain a peptide. The purified peptide thus obtained was supplied from Amersham Biosciences, Inc. “AKTA explorer10XT” [Column: Millipore, Inc. “NOVAPACK C18” 3.9 mmφ × 150 mm, mobile phase: acetonitrile and water containing 0.05% by volume of trifluoroacetic acid Of the mixed solvent (the acetonitrile concentration was linearly changed from 5 vol% to 50 vol% over 30 minutes) and a flow rate of 1.0 mL / min] showed a single peak at a retention time of 11.4 minutes. It was done. The molecular weight of the purified peptide determined by FAB mass spectrum was 2722 (theoretical value: 2722.9).

Dissolve 2.5 mg (0.0009 mmol) of H- (Pro-Hyp-Gly) ₄ -Glu-Hyp- (Pro-Hyp-Gly) ₅ -OH in 0.25 mL of 10 mM phosphate buffer (pH 7.4). And stirred at room temperature. To this mixture, 0.12 mg (0.0009 mmol) of 1-hydroxybenzotriazole, 1.7 mg (0.0092 mmol) of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride were added, and further 20 ° C. And stirring was continued for 2 days. The obtained reaction solution was diluted 10-fold with water and dialyzed against water for 3 days to remove reagents such as a condensing agent and unreacted monomers to obtain the polypeptide of the present invention. The ratio of peptide units (1) and (2) ((1) / (2)) was 90/10 (molar ratio).

  The obtained polypeptide was subjected to gel permeation chromatography (manufactured by Amersham Biosciences, AKTApurifier system, column: Superose 6 HR GL, flow rate: 0.5 mL / min, eluent: 10 mM phosphate buffer containing 150 mM NaCl ( When subjected to pH 7.4)), a polypeptide peak was observed in the molecular weight range of 100,000 to 2 million. The molecular weight was calculated using a Gel Filtration HMW Calibration Kit manufactured by Amersham Biosciences as a standard substance.

  When the circular dichroism spectrum of the obtained polypeptide was measured, a positive cotton effect was observed at 225 nm and a negative cotton effect was observed at 197 nm, confirming the formation of a triple helical structure.

Example 2
1.2 mg (0.00045 mmol) of formula: peptide represented by H- (Pro-Hyp-Gly) ₁₀ -OH (SEQ ID NO: 2) (Peptide Institute, Inc.) and 1.2 mg (0.00045 mmol) of Examples The peptide represented by the formula obtained in 1: H- (Pro-Hyp-Gly) ₄ -Glu-Hyp-Gly- (Pro-Hyp-Gly) ₅ -OH (SEQ ID NO: 1) is converted to 0.25 mL of 10 mM phosphorus. 0.12 mg (0.0009 mmol) 1-hydroxybenzotriazole, 15.7 mg (0.082 mmol) 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride, dissolved in an acid buffer (pH 7.4) The mixture was added and stirring was continued at 20 ° C. for 2 days. The obtained reaction solution was diluted 10-fold with water and dialyzed against water for 3 days to remove reagents such as a condensing agent and unreacted monomers to obtain the polypeptide of the present invention. The ratio of peptide units (1) and (2) ((1) / (2)) was 95/5 (molar ratio).

  The obtained polypeptide was subjected to gel permeation chromatography (manufactured by Amersham Biosciences, AKTApurifier system, column: Superose 6 HR GL, flow rate: 0.5 mL / min, eluent: 10 mM phosphate buffer containing 150 mM NaCl ( When subjected to pH 7.4)), a polypeptide peak was observed in the molecular weight range of 140,000 to 2,000,000. The molecular weight was calculated using a Gel Filtration HMW Calibration Kit manufactured by Amersham Biosciences as a standard substance.

  When the circular dichroism spectrum of the obtained polypeptide was measured, a positive cotton effect was observed at 224 nm and a negative cotton effect was observed at 196 nm, confirming the formation of a triple helical structure.

Comparative Example 1
A peptide represented by 2.4 mg (0.0009 mmol) of formula: H- (Pro-Hyp-Gly) ₁₀ -OH (SEQ ID NO: 2) (Peptide Institute, Inc.) was suspended in 1 mL of dimethyl sulfoxide, and at room temperature. Stir. To this mixture, 0.12 mg (0.0009 mmol) diisopropylethylamine, 0.12 mg (0.0009 mmol) 1-hydroxybenzotriazole, 0.34 mg (0.0018 mmol) 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide Hydrochloric acid was added and stirring was continued at 20 ° C. for 2 days. The resulting reaction solution was diluted 3 times with water and dialyzed against water for 3 days to remove reagents such as a condensing agent and unreacted monomers to obtain a polypeptide. The ratio of peptide units (1) and (2) ((1) / (2)) was 100/0 (molar ratio).

  The obtained polypeptide was subjected to gel permeation chromatography (manufactured by Amersham Biosciences, AKTApurifier system, column: Superose 6 HR GL, flow rate: 0.5 mL / min, eluent: 10 mM phosphate buffer containing 150 mM NaCl ( When subjected to pH 7.4)), a polypeptide peak was observed in the molecular weight range of 140,000 to 600,000. The molecular weight was calculated using a Gel Filtration HMW Calibration Kit manufactured by Amersham Biosciences as a standard substance.

  When the circular dichroism spectrum of the obtained polypeptide was measured, a positive cotton effect was observed at 224 nm and a negative cotton effect was observed at 196 nm, confirming the formation of a triple helical structure.

Example 3
One volume of an aqueous solution prepared by adjusting the polypeptide obtained in Examples 1 and 2 and the polypeptide obtained in Comparative Example 1 to a concentration of about 0.5 mg / ml was added to Na ⁺ 213 mM, K ⁺ 7.5 mM, Ca ²⁺ 3.8 mM, Mg ²⁺ 2.3 mM, Cl ⁻ 223.3 mM, HCO ₃ ⁻ 6.3 mM, HPO ₄ ²⁻ 1.5 mM, SO ₄ ²⁻ 0.75 mM, pH adjusted with Tris buffer after mixing with adjusted aqueous solution 2 ml to 7.4 (final ^{concentration, Na + 142mM, K + 5mM} , Ca 2+ 2.5mM, Mg 2+ 1.5mM, C1 - 148.8mM, HCO 3 - 4. 2mM, HPO ₄ ^2- 1mM, the SO ₄ ^2- 0.5mM), was immersed for 1 day at 36.5 ° C.. During this time, a small amount of solution is collected at any time, passed through a PD-10 column manufactured by Amersham Biosciences, salt removed, and then negatively stained with phosphotungstic acid, with a transmission electron microscope (Hitachi, H-7600) Observed.

  In the polypeptide obtained in Example 1, a fibrous aggregate structure having a diameter of about 10 nm and apatite crystals deposited on the surface were observed after 24 hours, as shown in FIG. A similar elephant was observed in the polypeptide of Example 2, but in the polypeptide of Comparative Example 1, a fibrous aggregate having a diameter of about 10 nm was observed, but no apatite precipitation was observed.

FIG. 1 is an electron micrograph of a complex of the polypeptide obtained in Example 1 and apatite (the scale bar in the figure indicates 100 nm).

Claims (13)

A novel chemically synthesized polypeptide comprising a peptide unit having an amino acid sequence represented by the following formula (1) and a peptide unit having an amino acid sequence represented by the following formula (2), and having a molecular weight of 5 × 10 ³ to 500 × shows the peak in the range of 10 ^4, chemically synthesized polypeptide can form at least a part of triple helical structure.
-Pro-X-Gly- (1)
-Y-Z-Gly- (2)
(Wherein X represents Pro or Hyp, Y represents Glu, and Z represents Hyp)   The polypeptide according to claim 1, wherein the ratio (molar ratio) between the peptide unit (1) and the peptide unit (2) is (1) / (2) = 99/1 to 1/99.   The polypeptide according to claim 1, wherein in a circular dichroism spectrum, a positive cotton effect is exhibited at a wavelength of 220 to 230 nm, and a negative cotton effect is exhibited at a wavelength of 195 to 205 nm. The polypeptide according to claim 1, which exhibits a peak in the molecular weight range of 1 × 10 ^{4 to} 500 × 10 ⁴ .   The polypeptide according to claim 1, which is capable of forming a collagen tissue.   The polypeptide according to claim 1, wherein apatites are supported.   An amino acid component or peptide fragment component comprising at least an amino acid or peptide fragment corresponding to formula (1) according to claim 1 and an amino acid or peptide fragment corresponding to formula (2) is condensed to form the chemistry according to claim 1. A method for producing a synthetic polypeptide.   A method for producing a chemically synthesized polypeptide having the amino acid sequence according to claim 1, comprising: (a) at least a peptide having both amino acid sequences represented by formulas (1) and (2) according to claim 1; A peptide component comprising, or (b) a peptide component comprising at least a peptide having the amino acid sequence represented by the formula (1) and a peptide having the amino acid sequence represented by the formula (2).   A method for producing a polypeptide on which the apatite is supported by bringing the chemically synthesized polypeptide of claim 1 into contact with an aqueous solution containing calcium ions and phosphate ions to deposit apatites on the polypeptide. The production method according to claim 9 , wherein the apatites are hydroxyapatite. A polypeptide carrying an apatite obtained by the production method according to claim 9 or 10 . A cosmetic material comprising a polypeptide, wherein the polypeptide is a chemically synthesized polypeptide according to claim 1 or 11 . A food additive comprising a polypeptide, wherein the polypeptide is a chemically synthesized polypeptide according to claim 1 or 11 .