Preparation of nano-apatite complex of silk-like materials with genetic engineering methods designed by detailed structural information on silk fibroins.

2005 Fiscal Year Final Research Report Summary

• Project/Area Number: 16350121
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Polymer/Textile materials
• Research Institution: National University Corporation Tokyo University of Agriculture and Technology
• Principal Investigator: ASAKURA Tetsuo Tokyo University of Agriculture and Technology Graduate School, Institute of Symbiotic Science and Technology, Professor, 大学院・共生科学技術研究部, 教授 (30139208)
• Co-Investigator(Kenkyū-buntansha): NISHIYAMA Norihiro Nihon University, School of dentistry at Matsudo, Professor, 松戸歯学部, 教授 (90112953)
• Project Period (FY): 2004 – 2005
• Keywords: Silk-like Protein / Solid state NMR Analysis / Biomaterials / Ca Binding Site / Lamella Structure / Peptide Solid Phase Synthesis / CalbindinD9K / Shell Nacreous Matrix Protein

Research Abstract

The purpose of this project is to develop new silk-like materials for regeneration of bone on the basis of the lamella structure of the crystalline domain of silk fibroins and selective Ca^<2+> binding to the turn region.
The generation of the lamella structure was proved for ^<13>C selectively -labeled (Ala-Gly)_<15> peptide which can be considered as the model of the crystalline domain using solid state NMR method. The stem part consisted of the repeated eight amino acid sequences. Several model peptides with silk sequences, (Ala)_<12>, (Ala-Gly)_6, (Ala-Gly-Gly)_4,(Ala-Gly-Gly-Gly)_3 and Ca^<2+> binding sites from Calbindin D_<9k> or Shell Nacreous Matrix Protein MSI60 were synthesized to study the character of new silk-like materials containing Ca^<2+> binding sites using solid state NMR.
In addition, the silk-like proteins containing Ca binding site from MSI60 with longer sequences were also prepared with genetic engineering method. The yield of the proteins prepared by the cultivation of E.coli was relatively high ; about 50 mg/l.
Ca binding to the surface of the proteins was performed by soaking the film of the protein in Ca-containing aqueous solvents alternatively. These silk-like proteins with hydroxyl apatite could be produced after these Ca binding experiments. These materials can be used for new silk-like materials for regeneration of bone.
These results have been published in the international Journals, Protein Science and Biomacromolecules.

Research Products

• [Journal Article] Conformational Study of Silk-like Peptides modified by the addition of the Calcium-binding Sequence from Shell Nacreous Matrix Protein MSI60 Using ^<13>C CP/MAS NMR Spectroscopy. (2006)
  • Author(s): Asakura, T., Hamada, M., Ha, S.-W., Knight,D.
  • Journal Title: Biomacromolecules (in press)
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Structural Analysis of Alanine Tripeptide with Anti-Parallel and Parallel β-sheet Structures in relation to the Analysis of Mixed β-Sheet Structures in Samia cynthia ricint Silk Fiber Using Solid State NMR. (2006)
  • Author(s): Asakura, T., Okonogi, M., Nakazawa, Y., Yamauchi,K.
  • Journal Title: J. Amer. Chem. Soc. (in press)
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Solid-State NMR Analysis of a Peptide (Gly-Pro-Gly-Gly-Ala)_6-Gly Derived from a Flagelliform Silk Sequence of Naphila clavipes. (2006)
  • Author(s): Ohgo, K., Kawase, T., Ashida, J., Asakura, T.
  • Journal Title: Biomacromolecules (in press)
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Biodegradation of Multilayer Silk Fibroin and Hydroxyapatite Composite Material. (2006)
  • Author(s): Kino, R., Ikoma, T., Yunoki, S., Monkawa, A., Matsuda, A., Kagata, G., Asakura, T., Munekata, M., Tanaka, J.
  • Journal Title: Key Engineeering Materials 309-311 Pages: 1169-1172
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Native Structure and Degradation Pattern of Silk Sericin Studied by ^<13>C NMR Spectroscopy. (2006)
  • Author(s): Teramoto, H., Kakazu, A., Asakura, T.
  • Journal Title: Macromolecules 39 Pages: 6-8
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Distinctive Influence of Two Hexafluoro Solvents on the Structural Stabilization of Bombyx mori Silk Fibroin Protein and Its Derived Peptides : ^<13>C NMR and CD Studies. (2006)
  • Author(s): Ha, S.-W., Asakura, T., Kishore, R.
  • Journal Title: Biomacromolecules 7 Pages: 18-23
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Structural Study of Silk-like Peptides Containing the Calcium-Binding Sequence from Calbindin D_<9k> Using ^<13>C CP/MAS NMR Spectroscopy. (2006)
  • Author(s): Asakura, T., Hamada, M., Nakazawa, Y., Ha, S.-W., Knight, D.
  • Journal Title: Biomacromolecule 7(2) Pages: 627-634
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Deposition of bone-like apatite on modified silk fibroin films from simulated body fluid. (2006)
  • Author(s): Kino, R., Ikoma, T., Monkawa, A., Yunoki, S., Munekata, M., Tanaka, J., Asakura, T.
  • Journal Title: J. Appl. Polym. Sci. 99 Pages: 2822-2830
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Conformational Study of Silk-like Peptides modified by the addition of the Calcium-binding Sequence from Shell Nacreous Matrix Protein MSI60 Using ^<13>C CP/MAS NMR Spectroscopy. (2006)
  • Author(s): Asakura, T., Hamada, M., Ha, S-W., Knight, D.
  • Journal Title: Biomacromolecules (in press)
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Structural Analysis of Alanine Tripeptide with Anti-Parallel and Parallelβ-sheet Structures in relation to the Analysis of Mixed β-Sheet Structures in Samia cynthia ricini Silk Fiber Using Solid State NMR. (2006)
  • Author(s): Asakura, T., Okonogi, M., Nakazawa, Y., Yamauchi, K.
  • Journal Title: J.Amer.Chem.Soc. (in press)
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Solid state NMR analysis of a peptide (Gly-Pro-Gly-Gly-Ala)_6-Gly derived from a flagelliform silk sequence of nephila clavipes. (2006)
  • Author(s): Ohgo, K., Kawase, T., Ashida, J., Asakura, T.
  • Journal Title: Biomacromolecules (in press)
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Biodegradation of Multilayer silk Fibroin and Hydroxyapatite Composite Material. (2006)
  • Author(s): Kino, R., Ikoma, T., Yunoki, S., Momkawa, A., Matsuda, A., Kagata, G., Asakura, T., Munekata, M., Tanaka, J.
  • Journal Title: Key Engineeering Materials 309-311 Pages: 1169-1172
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Native Structure and Degradation Pattern of Silk Sericin Studied by ^<13>C NMR Spectroscopy. (2006)
  • Author(s): Teramoto, H., Kakazu, A., Asakura, T.
  • Journal Title: Macromolecules 39(1) Pages: 6-8
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Distinctive Influence of Two Hexafluoro Solvents on the Structural Stabilization of Bombyx mori Silk Fibroin Protein and Its Derived Peptides : ^<13>C NMR and CD Studies. (2006)
  • Author(s): Ha, S.-W., Asakura, T., Kishore, R.
  • Journal Title: Biomacromolecules 7(1) Pages: 18-23
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Deposition of bone-like apatite on modified silk fibroin films from simulated body fluid. (2006)
  • Author(s): Kino, R., Ikoma, T., Monkawa, A., Yunoki, S., Munekata, M., Tanaka, J., Asakura, T.
  • Journal Title: J.Appl.Polym.Sci. 99 Pages: 2822-2830
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Possible Implications of Serine and Tyrosine Residues and Intermolecular Interactions on the Appearance of Silk I Structure of Bombyx mori Silk Fibroin-Derived Synthetic Peptides : High-Resolution ^<13>C Cross-Polarization/Magic-Angle Spinning NMR Study. (2005)
  • Author(s): Asakura, T., Ohgo, K., Ishida, T., Taddei, P., Monti, P., Kishore, R.
  • Journal Title: Biomacromolecules 6 Pages: 468-474
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] ^<13>C Solid-State NMR Study of Structural Heterogeneity in Peptides Containing Both Polyalanine and Repeated GGA Sequences as a Local Structural Model of Nephila clavipes Dragline Silk (Spidroin 1). (2005)
  • Author(s): Asakura, T., Yang, M., Kawase, T., Nakazawa, Y.
  • Journal Title: Macromolecules 38 Pages: 3356-3363
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Structural Determination of an Elastin-Mimetic Model Peptide, (Val-Pro-Gly-Val-Gly)6, Studied by ^<13>C CP/MAS NMR Chemical Shifts, Two-Dimensional off Magic Angle Spinning Spin-Diffusion NMR, Rotational Echo Double Resonance, and Statistical Distribution of Torsion Angles from Protein Data Bank. (2005)
  • Author(s): Ohgo, K., Ashida, J., Kumashiro, K.K., Asakura, T.
  • Journal Title: Macromolecules 38 Pages: 6038-6047
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Design, expression and solid-state NMR characterization of silk-like materials constructed from sequences of spider silk, Samia cynthia ricini and Bombyx mori silk fibroins. (2005)
  • Author(s): Yang, M., Asakura, T.
  • Journal Title: J. Biochemistry 137 Pages: 721-729
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Refinement of Repeated □-turn Structure for Silk I Conformation of Bombyx mori Silk Fibroin Using ^<13>C Solid-State NMR and X-ray Diffraction Methods. (2005)
  • Author(s): Asakura, T., Ohgo, K., Komatsu, K., Kanenari, M., Okuyama, K.
  • Journal Title: Macromolecules 38 Pages: 7397-7403
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Structure of Model Peptides Based on Nephila clavipes Dragline Silk Spidroin (MaSp1) Studied by ^<13>C Cross Polarization/Magic Angle Spinning NMR. (2005)
  • Author(s): Yang, M., Nakazawa, Y., Yamauchi, K., Knight, D., Asakura, T.
  • Journal Title: Biomacromolecules 6 Pages: 3220-3226
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Evidence from ^<13>C solid-state NMR spectroscopy for a lamella structure in an alanine-glycine copolypeptide : A model for the crystalline domain of Bombyx mori silk fiber. (2005)
  • Author(s): Asakura, T., Nakazawa, Y., Ohnishi, E., Moro, F.
  • Journal Title: Protein Science 14 Pages: 2654-2657
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Use of Microcoil Probehead for Determination of the Structure of Oriented Silk Fibers by Solid-State NMR. (2005)
  • Author(s): Yamauchi, K, Imada, T., Asakura, T.
  • Journal Title: J. Phys. Chem. B. 109 Pages: 17689-17692
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] ^<13>C Solid-State NMR Study of Structural Heterogeneity in Peptides Containing Both Polyalanine and Repeated GGA Sequences as a Local Structural Model of Nephila clavipes Dragline Silk (Spidroin 1). (2005)
  • Author(s): Asakura, T., Yang, M., Kawase, T.Nakazawa, Y.
  • Journal Title: Macromolecules 38 Pages: 3356-3363
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Structural Determination of an Elastin-Mimetic Model Peptide, (Val-Pro-Gly-Val-Gly)6, Studied by ^<13>C CP/MAS NMR Chemical Shifts, Two-Dimensional off Magic Angle Spinning Spin-Diffusion NMR, Rotational Echo Double Resonance, and Statistical Distribution of Torsion Angles from Protein Data Bank. (2005)
  • Author(s): Ohgo, K., Ashida, J., Kumashiro, K.K, Asakura, T.
  • Journal Title: Macromolecules 38 Pages: 6038-6047
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Design, expression and solid-state NMR characterization of silk-like materials constructed from sequences of spider silk, Samia cynthia ricini and Bombyx mori silk fibroins. (2005)
  • Author(s): Yang, M., Asakura, T.
  • Journal Title: J.Biochemistry 137 Pages: 721-729
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Refinement of Repeated β-turn Structure for Silk I Conformation of Bombyx mori Silk Fubroin Using ^<13>C Solid-State NMR and X-ray Diffraction Methods. (2005)
  • Author(s): Asakura, T., Ohgo, K., Komatsu, K., Kanenari, M., Okuyama, K.
  • Journal Title: Macromolecules 38 Pages: 7397-7403
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Structure of Model Peptides Based on Nephila clavipes Dragline Silk Spidroin (MaSp1) Studied by ^<13>C Cross Polarization/Magic Angle Spinning NMR. (2005)
  • Author(s): Yang, M., Nadazawa, Y., Yamauchi, K., Knight, D., Asakura, T.
  • Journal Title: Biomacromolecules 6 Pages: 3220-3226
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Evidence from 13C solid-state NMR spectroscopy for a lamella structure in an alanine-glycine copolypeptide : A model for the crystalline domain of Bombyx mori silk fiber. (2005)
  • Author(s): Asakura, T., Nakazawa, Y., Ohnishi, E., Moro, F.
  • Journal Title: Protein Science 14 Pages: 2654-2657
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Use of Microcoil Probehead for Determination of the Structure of Oriented Silk Fibers by Solid-State NMR. (2005)
  • Author(s): Yamauchi, K., Imada, T., Asakura, T.
  • Journal Title: J.Phys.Chem.B. 109 Pages: 17689-17692
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Production and characterization of a silk-like hybrid protein, based on the polyalanine region of Samia cynthia ricini silk fibroin and a cell adhesive region derived from fibronectin. (2004)
  • Author(s): Asakura, T., Tanaka, C., Yang, M., Yao, J., Kurokawa, M.
  • Journal Title: Biomaterials 25 Pages: 617-624
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Structure of the Model Peptides of Bombyx mori Silk-Elastin Like Protein studied with Solid State NMR. (2004)
  • Author(s): Ohgo, K., Kurano Tracie, L., Kumashiro Kristin, K., Asakura, T.
  • Journal Title: Biomacromolecules 5 Pages: 744-750
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Ulrich Anne, S., Structural role of tyrosine in Bombyx mori silk fibroin, studied by solid-state NMR and molecular mechanics on a model peptide prepared as silk I and II. (2004)
  • Author(s): Asakura, T., Suita, K., Kameda, T., Afonin, S.
  • Journal Title: Magnetic resonance in chemistry 42 Pages: 258-266
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Structures of Bombyx mori and Samia cynthia ricini Silk Fibroins Studied with Solid-State NMR. (2004)
  • Author(s): Yao, J., Nakazawa, Y., Asakura, T.
  • Journal Title: Biomacromolecules 5 Pages: 680-688
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Spectroscopic Characterization of Heterogeneous Structure of Samia Cynthia ricini Silk Fibroin Induced by Stretching and Molecular Dynamics Simulation. (2004)
  • Author(s): Yang, M., Yao, J., Sonoyama, M., Asakura, T.
  • Journal Title: Macromolecules 37 Pages: 3497-3504
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Structural Analysis of Bombyx mori silk fibroin peptides with formic acid treatment using high-resolution solid-state 13C NMR spectroscopy. (2004)
  • Author(s): Yao, J., Ohgo, K., Sugino, R., Kishore, R., Asakura, T.
  • Journal Title: Biomacromolecules 5 Pages: 1763-1769
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Study of protein conformation and orientation in silkworm and spider silk fibers using Raman microspectroscopy. (2004)
  • Author(s): Rousseau, M.-E., Lefevre, T., Beaulieu, L., Asakura, T., Pezolet, M.
  • Journal Title: Biomacromolecules 5 Pages: 2247-2257
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Raman study of poly(alanine-glycine)-based peptides containing tyrosine, valine, and serine as model for the semicrystalline domains of Bombyx mori silk fibroin. (2004)
  • Author(s): Taddei, P., Asakura, T., Yao, J., Monti, P.
  • Journal Title: Biopolymers 75 Pages: 314-324
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Structure of characteristic sequences in Nephila clavipes dragline silk (MaSp1) studied with ^<13>C solid state NMR. (2004)
  • Author(s): Asakura, T., Yang, M., Kawase, T.
  • Journal Title: Polymer Journal 36 Pages: 999-1003
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Design, expression and characterization of collagen-like proteins based on the cell adhesive and crosslinking sequences derived from native collagens. (2004)
  • Author(s): Yao, J., Yanagisawa, S., Asakura, T.
  • Journal Title: Journal of Biochemistry 136 Pages: 643-649
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Structural role of tyrosine in Bombyx mori silk fibroin, studied by solid-state NMR and molecular mechanics on a model peptide prepared as slik I and II. (2004)
  • Author(s): Asakura, T., Suita, K., Kameda, T., Afonin, S., Ulrich Anne, S.
  • Journal Title: Magnetic resonance in chemistry 42 Pages: 258-266
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Structures of Bombyx mori and Samia cynthia ricini silk Fibroins Studied with Solid-State NMR. (2004)
  • Author(s): Yao, J., Nakazawa, Y., Asakura, T.
  • Journal Title: Biomacromolecules 5 Pages: 680-688
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Study of protein conformation and orientation in silk worm and spider silk fibers using Raman microspectroscopy. (2004)
  • Author(s): Rousseau, M.-E., Lefevre, T., Beaulieu, L., Asakura, T., Pezolet, M.
  • Journal Title: Biomacromolecules 5 Pages: 2247-2257
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Raman study of poly(alanine-glycine)-based peptides contatining tyrosine, valine, and serine as model for the semicrystalline domains of Bombyx mori silk fibroin. (2004)
  • Author(s): Taddei, P., Asakura, T., Yao, J., Monti, P.
  • Journal Title: Biopolymers 75 Pages: 314-324
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Production of interferon-beta by fibroblast cells on membranes prepared with RGD-containing peptides. (2004)
  • Author(s): Higuchi, A., Takanashi, Y., Tsuzuki, N., Asakura, T., Cho, C.-S., Akaike, T., Hara, M.
  • Journal Title: J Biomed Mater Res A 65 Pages: 369-378
  • Description: 「研究成果報告書概要(欧文)」より
• [Book] 「エレクトロスピニング法」機能性不織布の新展開 (2004)
  • Author(s): 大郷耕輔, 朝倉哲郎
  • Total Pages: 235
  • Publisher: シーエムシー出版
  • Description: 「研究成果報告書概要(和文)」より