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2002 Fiscal Year Final Research Report Summary

Development of high efficiency cell-free protein production and purification system

Research Project

Project/Area Number 13650830
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field 反応・分離工学
Research InstitutionKobe University

Principal Investigator

KONDO Akihiko  Kobe University, Faculty of Engineering, Professor, 工学部, 教授 (40205547)

Project Period (FY) 2001 – 2002
Keywordsmlecular chaperon / foldase / fine particles / cell-free protein synthesys / stimuli-sensitive polymer / protein / folding / affinity separation
Research Abstract

In this study, efficient system for cell-free synthesis of proteins from various cDNA and affinity purification was developed. To improve the efficiency of cell-free protein synthesis system, we compared the two phase system and dialysis system. By adopting dialysis system, the final amount of synthesized protein increased significantly. Then, we established the efficient production system of molecular chaperons and foldases, and their effects on the protein folding were investigated. By combining chaperonin and disulfide isomerase (Dsb protein) from Escherichia coli, protein refolding efficiency was improved significantly. We have also developed thermo-responsive magnetic nanoparticles for immobilization of chaperonin and foldases and affinity separation. The thermno-responsive magnetic nanoparticles (average diameter of 100 nm) were synthesized by coating magnetite nanoparticles (average diameter of 10-20 nm) with a thermoresponsive polymer consisting of N-acryloilglycine amide (NAGAm) and biotin derivative. These magnetic nanoparticles showed a reversible transition between flocculation and dispersion at around upper critical flocculation temperature. The thermo-responsive magnetic nanoparticles were proved to be effective for immobilization of molecular chaperones and foldases and affinity separation of synthesized proteins.

  • Research Products

    (10 results)

All Other

All Publications (10 results)

  • [Publications] Jiro Kohda: "Improvement of productivity of active form of glutamate racemase in escherichia coli by coexpression of folding accessory proteins"Biochemical Engineering Journal. 10. 39-45 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Jiro Kohda: "Effect of oxidized and reduced forms of escherichia coli DsbC on protein refolding"Journal of Bioscience and Bioengineering. 94. 130-134 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 近藤 昭彦: "磁性ナノ微粒子材料の開発とバイオ・ナノテクへの展開、未来材料"未来材料. 2. 19-25 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Jiro Kohda: "Refolding of proteins by hexadecamers and monomers of and subunits of group II chaperonin from hyperthermophilic archaeum, thermococcus strain KS-1"Biochemical Engineering Journal. (in press).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hrotaka Furukawa: "Affinity selection of target cells from cell surface displayed libraries : A nobel procedure using thermo-responsive magnetic nanoparticles"Applied Microbiology and Biotechnology. (in press).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Jiro Kohda: "Improvement of productivity of active form of glutamate racemase in escherichia coli by coexpression of folding accessory proteins"Biochemical Engineering Journal. 10(1). 39-45 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Jiro Kohda: "Effect of oxidized and reduced forms of escherichia coli DsbC on protein refolding"Journal of Bioscience and Bioengineering. 94(2). 130-134 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Akihiko Kondo: "Development of magnetic nanoparticles and their application to bionano-technology"Expected Materials for the Future. 2(10). 19-25 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Jiro Kohda: "Refolding of proteins by hexadecamers and monomers of and subunits of group II chaperonin from hyperthermophilic archaeum, thermococcus strain KS-1"Biochemical Engineering Journal. in press.

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hirotaka Furukawa: "Affinity selection of target cells from cell surface displayed libraries : A nobel procedure using thermo-responsive magnetic nanoparticles"Applied Microbiology and Biotechnology. in press.

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 2004-04-14  

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