WO2005078085A1 - コラーゲン様構造を有するポリペプチド - Google Patents
コラーゲン様構造を有するポリペプチド Download PDFInfo
- Publication number
- WO2005078085A1 WO2005078085A1 PCT/JP2005/001583 JP2005001583W WO2005078085A1 WO 2005078085 A1 WO2005078085 A1 WO 2005078085A1 JP 2005001583 W JP2005001583 W JP 2005001583W WO 2005078085 A1 WO2005078085 A1 WO 2005078085A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- peptide
- pro
- trimer
- gly
- hyp
- Prior art date
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 143
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 33
- 229920001184 polypeptide Polymers 0.000 title abstract description 12
- 239000013638 trimer Substances 0.000 claims abstract description 58
- 238000000034 method Methods 0.000 claims abstract description 13
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 10
- 239000000539 dimer Substances 0.000 claims description 12
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 125000006239 protecting group Chemical group 0.000 claims description 5
- 230000003213 activating effect Effects 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 abstract description 7
- 102000008186 Collagen Human genes 0.000 description 24
- 108010035532 Collagen Proteins 0.000 description 24
- 229920001436 collagen Polymers 0.000 description 24
- 230000015572 biosynthetic process Effects 0.000 description 14
- 239000000243 solution Substances 0.000 description 13
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 12
- 238000004128 high performance liquid chromatography Methods 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 238000005259 measurement Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000000108 ultra-filtration Methods 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- 238000001142 circular dichroism spectrum Methods 0.000 description 4
- 239000000470 constituent Substances 0.000 description 4
- 239000012299 nitrogen atmosphere Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 239000011550 stock solution Substances 0.000 description 4
- WTKQMHWYSBWUBE-UHFFFAOYSA-N (3-nitropyridin-2-yl) thiohypochlorite Chemical compound [O-][N+](=O)C1=CC=CN=C1SCl WTKQMHWYSBWUBE-UHFFFAOYSA-N 0.000 description 3
- VOUAQYXWVJDEQY-QENPJCQMSA-N 33017-11-7 Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)NCC(=O)NCC(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N1[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)CCC1 VOUAQYXWVJDEQY-QENPJCQMSA-N 0.000 description 3
- 108010075254 C-Peptide Proteins 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 238000010647 peptide synthesis reaction Methods 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 102100035846 Pigment epithelium-derived factor Human genes 0.000 description 2
- 102000029797 Prion Human genes 0.000 description 2
- 108091000054 Prion Proteins 0.000 description 2
- -1 acetamidomethyl Chemical group 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 239000000560 biocompatible material Substances 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- RLSSMJSEOOYNOY-UHFFFAOYSA-N m-cresol Chemical compound CC1=CC=CC(O)=C1 RLSSMJSEOOYNOY-UHFFFAOYSA-N 0.000 description 2
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- 108090000102 pigment epithelium-derived factor Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- UQDJGEHQDNVPGU-UHFFFAOYSA-N serine phosphoethanolamine Chemical compound [NH3+]CCOP([O-])(=O)OCC([NH3+])C([O-])=O UQDJGEHQDNVPGU-UHFFFAOYSA-N 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 230000019635 sulfation Effects 0.000 description 2
- 238000005670 sulfation reaction Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 238000002834 transmittance Methods 0.000 description 2
- KLBPUVPNPAJWHZ-UMSFTDKQSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)SC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KLBPUVPNPAJWHZ-UMSFTDKQSA-N 0.000 description 1
- CSMYOORPUGPKAP-IBGZPJMESA-N (2r)-3-(acetamidomethylsulfanyl)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CSCNC(=O)C)C(O)=O)C3=CC=CC=C3C2=C1 CSMYOORPUGPKAP-IBGZPJMESA-N 0.000 description 1
- WPBXBYOKQUEIDW-VFNWGFHPSA-N (2s,4r)-1-(9h-fluoren-9-ylmethoxycarbonyl)-4-[(2-methylpropan-2-yl)oxy]pyrrolidine-2-carboxylic acid Chemical compound C1[C@H](OC(C)(C)C)C[C@@H](C(O)=O)N1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 WPBXBYOKQUEIDW-VFNWGFHPSA-N 0.000 description 1
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- DHDOADIPGZTAHT-YUMQZZPRSA-N Gly-Glu-Arg Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DHDOADIPGZTAHT-YUMQZZPRSA-N 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- VKZGJEWGVNFKPE-UHFFFAOYSA-N N-Isobutylglycine Chemical group CC(C)CNCC(O)=O VKZGJEWGVNFKPE-UHFFFAOYSA-N 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- 101100163901 Rattus norvegicus Asic2 gene Proteins 0.000 description 1
- 239000002262 Schiff base Substances 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- HAXFWIACAGNFHA-UHFFFAOYSA-N aldrithiol Chemical compound C=1C=CC=NC=1SSC1=CC=CC=N1 HAXFWIACAGNFHA-UHFFFAOYSA-N 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000000429 assembly Methods 0.000 description 1
- 230000000712 assembly Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 125000002228 disulfide group Chemical group 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
- 238000005734 heterodimerization reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
- C07K5/0823—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp and Pro-amino acid; Derivatives thereof
Definitions
- the present invention relates to a polypeptide having a collagen-like triple helical structure, a peptide trimer for producing such a polypeptide, and a method for producing a polypeptide and a peptide trimer.
- Collagen is widely used as a base material for pharmaceuticals and cosmetics, a biocompatible material for regenerative medicine and drug delivery systems, a support for tissue culture, and the like.
- the collagen currently in use is a refined version of collagen that has also gained animal power, mainly from pigs and pests.
- the risk of prion infection causing BSE is a problem.
- collagen derived from plants and fish skin has come to be used. Therefore, there is a need for a safe supply of collagen substitutes.
- efforts to construct a recombinant human collagen-producing system using various host organisms have not been realized yet.
- Collagen has a structure in which three polypeptide chains form a long three helix. It is well known that three chemically synthesized peptides having a collagen-like sequence self-assemble to form the same three-helical structure as collagen, and have been used for structural studies of collagen.
- the collagen-like polypeptide include a peptide unit [-(Pro-Y-Gly) n-] a (where Y represents Pro or Hyp, and n represents an integer of 1 to 20) and a peptide unit. [-(Z) r-] b (wherein, Z represents a peptide chain having 1 to 10 amino acid residues, and r represents an integer of 1 to 20).
- Supramolecules that also have the ability to sequence collagen-like peptides include the collagen triblock peptide of Martin et al .: (Glu) (Gly-X-Hyp-Gly-Pro-Hyp)
- An object of the present invention is to provide a polypeptide having a collagen-like triple helical structure and a peptide unit for producing such a polypeptide.
- the present invention provides a compound represented by the formula:
- the present invention provides a peptide trimer in which three peptides having the same chain length and having the same repeating unit as a basic structure are bonded to be shifted from each other in the main chain direction.
- the three peptides are connected to each other by a disulfide bond.
- the present invention provides a method for producing the above-described peptide trimer of the present invention. This method
- a first peptide having one Cys, a second peptide having two Cys, one of which has a protected SH group, and a third peptide having one Cys are provided. Forming a peptide dimer by bonding the peptide of the second peptide and the second peptide by a disulfide bond,
- the present invention provides a molecular assembly having a triple helical structure, comprising the above-described peptide trimer of the present invention as a constituent.
- the present invention further provides a method for producing the molecular assembly of the present invention, wherein the solution of the peptide trimer is left at a temperature in the range of 0 ° C to 40 ° C for 1 hour or more. And provide a method.
- FIG. 1 is a schematic diagram of a peptide trimer of the present invention and a molecular assembly having a collagen-like three helix comprising the same as a constituent unit.
- FIG. 2 shows various structures of the peptide trimer of the present invention.
- FIG. 3 shows an example of the amino acid sequence of a peptide trimer of the present invention and a molecular assembly comprising the same as a constituent unit.
- FIG. 4 shows the results of measuring circular dichroism spectra of molecular assemblies prepared according to the present invention.
- FIG. 5 shows the ultrafiltration membrane permeability of a molecular assembly prepared according to the present invention.
- FIG. 6 shows another embodiment of the peptide trimer of the present invention.
- FIG. 7 shows the particle size distribution of a molecular assembly prepared according to the present invention.
- the present invention provides a peptide trimer unit formed by trimerizing a synthetic peptide having a collagen-like sequence by regioselective crosslinking.
- the present invention also provides a method for preparing a supramolecule having a triple helical structure using the peptide trimer unit as a constituent unit. An overview of the present invention is shown in FIG.
- the peptide trimer unit of the invention has the formula:
- the polypeptide chain that constitutes natural collagen has a repeating unit of-(-Gly-Pro-Pro-)-or-(-Gly-Pro-Hyp-)-as its basic structure, and has a long left-handed spiral.
- the three polypeptide chains can wrap around each other to form a long right-handed helical structure due to hydrogen bonding between the chains.
- leaving a solution of a peptide with a chain length of about 15-30 having repeating units of-(-Gly- Pro- Pro-)-or-(-Gly- Pro- Hyp-)- It is known to have a stable three-helix structure similar to collagen. In the present specification, such a triple helical structure is referred to as “collagen-like structure”.
- the peptide trimer unit of the present invention is composed of three peptides, and each peptide is represented by-(-Gly-XY-)-where X and Y are any amino acid residues ] Represents a repeating unit.
- X and Y are equivalent to natural amino acid residues.
- the amino acid residue may be a modified amino acid residue well known in the art, and may be an L-form or a D-form.
- the peptide trimers of the invention are found in natural collagen
- Peptide ⁇ has a basic structure that also has a-(-Gly-XY-)-repeating unit force '' means that all amino acid sequences of the peptide have-(-Gly-XY-)-repeating units ( That is, it has Gly for every third), which means that it may include a part that does not have such a repeating unit.
- 80% or more, preferably 90% or more of the amino acid sequences in the peptide trimer molecule have-(-Gly-XY-)-repeating units. It is necessary to be.
- the peptide trimer unit of the present invention has a structure in which three peptides having the same chain length are shifted from each other in the main chain direction and amino acid side chains are bonded to each other.
- “shifted from each other in the main chain direction” means that three peptides are bonded so that they do not completely overlap with each other.
- various types of peptides shown in FIG. Structure is conceivable.
- the single-chain or double-chain portion in the peptide trimer plays a marginal role to stably hold a plurality of peptide trimers by hydrogen bonding. Therefore, in the present invention, among the structures shown in FIG. 2, a structure in which the single-stranded or double-stranded region is long and the triple-stranded region is short is more preferable.
- Each peptide constituting the peptide trimer unit of the present invention has the same chain length. As a result, when the peptide trimers assemble to form a triple helical structure, a stable structure without gaps can be obtained.
- Each peptide can be synthesized by a known peptide synthesis method (liquid phase method or solid phase method). Chain length is 10-60 amino acid residues, preferably Is 15-40 residues, more preferably 20-30 residues. If the chain length is short, the stability of the three-stranded structure formed will be low, and if the chain length is long, the cost of peptide synthesis will increase.
- the amino acid side chains of three peptides are preferably bonded.
- the three peptides have a Cys at the appropriate position and the peptides are linked together by a disulfide bond.
- amino acid side chains may be covalently bonded to each other by an amide bond, a sulfide bond, a Schiff base bond, or the like.
- the peptide trimer unit of the present invention is prepared by a site-specific disulfide bond formation method (Ottl, Jetal, FEBS Lett, 398, 31-36, 1996; Ottl, J. and Moroder, L., J. Am. Chem. Soc. 121, 653-661, 1999; Koide, T. et al. Bioorg Med Chem Lett. 14, 125-128, 2004) to form an interchain disulfide bond.
- a site-specific disulfide bond formation method Ottl, Jetal, FEBS Lett, 398, 31-36, 1996; Ottl, J. and Moroder, L., J. Am. Chem. Soc. 121, 653-661, 1999; Koide, T. et al. Bioorg Med Chem Lett. 14, 125-128, 2004
- the protective group for the SH group any protective group known in the field of peptide synthesis may be used, and for example, acetamidomethyl can be used.
- the first peptide and the second peptide are linked by a disulfide bond to form a peptide dimer.
- Methods for linking the SH groups of Cys via disulfide bonds are generally known in the art, and include, for example, pyridine sulfylation or -tropyridine sulfalelation of the SH group on one peptide chain.
- a peptide dimer in which Cys residues are disulfide bonded to each other can be formed.
- the protected SH of the second peptide of the peptide dimer is activated by conversion of a protecting group, and the peptide dimer and the third peptide are similarly bound by a disulfide bond.
- the peptide trimer of the present invention can be obtained.
- the peptide trimer of the present invention may be purified by column chromatography, HPLC, etc., if necessary. You can do it.
- the collagen-like molecular assembly (supramolecule) of the present invention has a fibrous structure having a triple helical structure similar to collagen using the peptide trimer unit of the present invention as its structural unit (FIGS. 1, 3). .
- a solution of the above-mentioned peptide trimer unit of the present invention is left at a low temperature, complementary self-assembly using the formation of a triple helix as a driving force occurs. That is, the molecular assembly of the present invention is obtained by treating the solution containing the above-mentioned peptide trimer unit of the present invention at 0 ° C to 40 ° C, preferably at 0 ° C at 10 ° C, for 1 hour or more, preferably for 12 hours.
- the film is left standing for 48 hours or more.
- An aqueous solution is preferable as the solution, but an organic solvent can be used because the collagen triple-helical structure does not have a hydrophobic core present in many other proteins.
- the strength of the molecular aggregate having a collagen-like three-helical structure is confirmed by analyzing the structure of the product by measuring circular dichroism spectrum, measuring the molecular weight, and directly observing with an electron microscope. be able to.
- the collagen-like molecular assembly of the present invention can be used in all fields where natural collagen is currently used. For example, it can be used as a cell culture base, a drug delivery system, and other biocompatible materials.
- the collagen-like molecular assembly of the present invention has the advantage that there is no danger of prion infection or gelatin allergy that causes BSE, which is currently a problem in the use of natural collagen.
- the collagen-like molecular assembly of the present invention can be used for functional analysis of collagen and development of functional artificial collagen.
- Collagen-like supramolecules having specific functions binding ability to specific protein binding
- COIDE method Yamamoto method
- PET method Yasui, N. and Koide, T "J. Am. Chem. Soc. 125, 15728-15729, 2003
- PEDF pigment epithelium-derived factor
- Solvent A, 0.05% trifluoroacetic acid Z water
- TrtA-PEG-Resin Wanganabe Chemical Industry
- Fmoc-Hyp (tBu) -OH As a side chain functional group protected amino acid, Fmoc-Hyp (tBu) -OH,
- Fmoc-Cys (Acm) -OH and Fmoc-Cys (Trt) -OH were used (abbreviation: Hyp or 0,4-hydroxyproline; Acm, acetamidomethyl; Trt, trityl).
- M-peptide (Acm, SH form) was dissolved in 50 mM sodium acetate (pH 5.4) [buffer A] containing 2 mM EDTA, and 2,2'-dipyridyl disulphide (49.5 mg, 225 ⁇ mol) was mixed with 2-propanol in a nitrogen atmosphere at room temperature and reacted for 1 hour.
- the produced target product was purified by HPLC and freeze-dried.
- the peptide trimer unit was dissolved in water at a concentration of 10 mg / ml and left at 4 ° C for 14 days (stock solution).
- the stock solution was diluted 5-fold with water at 4 ° C and used for the measurement.
- the measurement conditions are as follows:
- FIG. 4 shows the results.
- the value of the average molar ellipticity of residues ([0] mrw) at 225 nm is equivalent to that of the native collagen and collagen model peptide triple helix. It is suggested that.
- the three-helical structure is changed by heat. And a random coil structure.
- the supramolecular stock solution or the same three-helix (Pro-Hyp-Gly) -amide at 4 ° C as a control was diluted 20-fold with water at 4 ° C.
- H-Hyp-Gly-Pro-Hyp-Gly-Cys (Acm) -Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Cys (SH) -Gly-Pro -Hyp-Gly-Pro-OH (SEQ ID NO: 5)
- a trimer was synthesized in the same manner as in Example 1, and mass spectrometry confirmed that the target compound was synthesized.
- the staggered trimer was supramolecularized at low temperature in the same manner as in Example 2, and the circular dichroism spectrum was measured. As a result, almost the same results as in Example 2 (FIG. 4) were obtained.
- a stock solution of the staggered trimer (10 mg ZmL) was diluted 12-fold with water at 4 ° C, and then the particle size distribution of the sample left at 4 ° C for 1 hour was measured. The measurement was performed using a Shimadzu laser diffraction particle size distribution analyzer (SALD-700) at 4 ° C and a laser wavelength of 405 nm.
- Fig. 7 shows the results. Supramolecular structures with particle sizes of about 0.5 and about 10 microns were detected.
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/589,557 US8030448B2 (en) | 2004-02-16 | 2005-02-03 | Polypeptide having collagen-like structure |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2004-038612 | 2004-02-16 | ||
JP2004038612 | 2004-02-16 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2005078085A1 true WO2005078085A1 (ja) | 2005-08-25 |
Family
ID=34857814
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2005/001583 WO2005078085A1 (ja) | 2004-02-16 | 2005-02-03 | コラーゲン様構造を有するポリペプチド |
Country Status (2)
Country | Link |
---|---|
US (1) | US8030448B2 (ja) |
WO (1) | WO2005078085A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010125722A1 (ja) * | 2009-05-01 | 2010-11-04 | 国立大学法人 千葉大学 | 人工コラーゲンを用いた軟骨培養用基材及び該基材を用いた軟骨再生治療方法 |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013090770A2 (en) * | 2011-12-14 | 2013-06-20 | The Board Of Trustees Of The University Of Arkansas | Delivery of therapeutic agents by a collagen binding protein |
WO2018148573A1 (en) | 2017-02-10 | 2018-08-16 | The Board Of Trustees Of The University Of Arkansas | Collagen-binding agent compositions and methods of using the same |
CN117903291A (zh) * | 2024-03-19 | 2024-04-19 | 如凤凰再生科技发展(成都)有限公司 | 一种三螺旋胶原蛋白及其制备方法和用途 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH10500298A (ja) * | 1994-05-16 | 1998-01-13 | メディカル リサーチ カウンシル | 三量体化ポリペプチド、その製造及び使用 |
JP2001514189A (ja) * | 1997-08-25 | 2001-09-11 | ウイスコンシン アラムニ リサーチ ファンデーション | コラーゲン擬似物 |
-
2005
- 2005-02-03 WO PCT/JP2005/001583 patent/WO2005078085A1/ja active Application Filing
- 2005-02-03 US US10/589,557 patent/US8030448B2/en not_active Expired - Fee Related
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH10500298A (ja) * | 1994-05-16 | 1998-01-13 | メディカル リサーチ カウンシル | 三量体化ポリペプチド、その製造及び使用 |
JP2001514189A (ja) * | 1997-08-25 | 2001-09-11 | ウイスコンシン アラムニ リサーチ ファンデーション | コラーゲン擬似物 |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010125722A1 (ja) * | 2009-05-01 | 2010-11-04 | 国立大学法人 千葉大学 | 人工コラーゲンを用いた軟骨培養用基材及び該基材を用いた軟骨再生治療方法 |
JPWO2010125722A1 (ja) * | 2009-05-01 | 2012-10-25 | 国立大学法人 千葉大学 | 人工コラーゲンを用いた軟骨培養用基材及び該基材を用いた軟骨再生治療方法 |
JP5704344B2 (ja) * | 2009-05-01 | 2015-04-22 | 国立大学法人 千葉大学 | 人工コラーゲンを用いた軟骨培養用基材及び該基材を用いた軟骨再生治療方法 |
Also Published As
Publication number | Publication date |
---|---|
US20070149764A1 (en) | 2007-06-28 |
US8030448B2 (en) | 2011-10-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Apostolovic et al. | Coiled coils: attractive protein folding motifs for the fabrication of self-assembled, responsive and bioactive materials | |
Fallas et al. | Synthetic collagen mimics: self-assembly of homotrimers, heterotrimers and higher order structures | |
Mart et al. | Peptide-based stimuli-responsive biomaterials | |
Lehrman | Protein structure | |
Voyer | The development of peptide nanostructures | |
IL190863A (en) | Method for manufacturing multilayer films and microcapsules comprising polypeptides | |
AU716531B2 (en) | Collagen-like peptoid residue-containing structures | |
WO2021167107A1 (ja) | ヒトトランスフェリンレセプター結合ペプチド | |
EP2764018B1 (en) | Self-assembling polypeptide polyhedra | |
WO2023165476A1 (zh) | 针对sort1的多肽化合物及其药物偶联物 | |
WO2005078085A1 (ja) | コラーゲン様構造を有するポリペプチド | |
Bellmann‐Sickert et al. | Palmitoylated SDF1 α shows increased resistance against proteolytic degradation in liver homogenates | |
JPH02500517A (ja) | ペプチド化合物 | |
JP5601437B2 (ja) | コラーゲン様構造を有するポリペプチド | |
Futaki et al. | Embodying a stable α-helical protein structure through efficient chemical ligation via thioether formation | |
JP2019535702A (ja) | ジンセンチド及びジンセンチド様ペプチドの調製及び使用 | |
EP1997826A2 (en) | Synthetic ligands for immunoglobulins and pharmaceutical compositions containing them | |
AU2014261381B2 (en) | Protransduzin B, a gene transfer enhancer | |
Mimna et al. | Toward the design of highly efficient, readily accessible peptide N-caps for the induction of helical conformations | |
CN116987152B (zh) | 一种结合沙贝冠状病毒s蛋白rbd结构域的环肽及其应用 | |
JP4448947B2 (ja) | ナノファイバー形成能を有するペプチド、及びペプチドナノファイバー | |
JPH10338700A (ja) | 新規ペプチド化合物 | |
Banerjee et al. | The chemical synthesis of α-conotoxins and structurally modified analogs with enhanced biological stability | |
Curtis | Hierarchical Assembly of Coiled-Coil Peptides: Protein Inclusion and Self-Replication | |
WO1989001943A1 (en) | Peptide complexes having enhanced stability |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWW | Wipo information: withdrawn in national office |
Country of ref document: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2007149764 Country of ref document: US Ref document number: 10589557 Country of ref document: US |
|
122 | Ep: pct application non-entry in european phase | ||
WWP | Wipo information: published in national office |
Ref document number: 10589557 Country of ref document: US |