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Development of rapid proteomic analysis for effective production of recombinant proteins by secretion

Research Project

Project/Area Number 13555226
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section展開研究
Research Field 生物・生体工学
Research InstitutionKobe University

Principal Investigator

KATOH Shigeo  Kobe University, Graduate School of Science and Technology, Professor, 自然科学研究科, 教授 (20026272)

Co-Investigator(Kenkyū-buntansha) TANAKA Hisao  Kyowa Hakko Kogyo Co., LTD., General Manager, 工務部, 技術開発室長
SHIOMI Naofumi  Kobe College, Dept. of Human Science, Associate Professor, 人間科学部, 助教授 (20299077)
Project Period (FY) 2001 – 2002
Project Status Completed (Fiscal Year 2002)
Budget Amount *help
¥13,900,000 (Direct Cost: ¥13,900,000)
Fiscal Year 2002: ¥4,200,000 (Direct Cost: ¥4,200,000)
Fiscal Year 2001: ¥9,700,000 (Direct Cost: ¥9,700,000)
Keywordsproduction by secretion / α-amylase / proteomic analysis / liposome / membrane blotting / methanol assimilating enzymes / 抗ペプチド抗体 / C-末端健全体 / プロテアーゼ
Research Abstract

Recombinant proteins have been produced by secretion from recombinant cells, since biologically active proteins are obtained through easy separation processes. However, the productivity of target proteins is not always high because of incorrect processing of signal sequences and degradation by proteases. In this work we propose an effective control and production method for secretion of recombinant proteins based on the proteomic analysis with rapid characterization of produced proteins and measurements of the activities of marker enzymes and proteases.
As a model protein α-amylase was secreted from S. cerevisiae and Pichia pastoris, and the effects of fermentation conditions on the productivity, the activities of marker enzymes and the characteristics of secreted protein were studied. The following results were obtained;
* Characteristics of C-terminus of secreted α-amylase were clarified by use of an anti-peptide antibody against the C-terminal sequences.
* Pichia pastoris could effectively secrete α-amylase by induction with methanol, and the feed of methanol could be controlled by the DO-statt for effective production.
* A novel membrane blotting method utilizing immunoliposomes was developed for rapid and highly sensitive measurement of proteins.
* The activities of enzymes in the metabolic pathway of methanol increased by induction with methanol and were related to the specific production rate of α-amylase.

Report

(3 results)
  • 2002 Annual Research Report   Final Research Report Summary
  • 2001 Annual Research Report
  • Research Products

    (13 results)

All Other

All Publications (13 results)

  • [Publications] 熊田 陽一: "Liposome Immunoblotting Assay Using a Substrate Forming Precipitate Inside Immunoliposomes"Biotechnology & Bioengineering. 30. 414-418 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] 黒川 朋世: "Secretion of α-Amylase from Saccharomyces cerevisiae and Pichia pastoris and Characterization of Its C-Terminus with an Anti-Peptide Antibody"Journal of Chemical Engineering of Japan. 35. 1277-1281 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Chunyeon Lee: "High Dissolved Oxygen Tension Enhances Heterologous Protein Expression by Recombinant Pichia pastoris"Process Biochem. (in press). (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Chunyeon Lee: "Effects of Substrate Feed Rates on Heterologous Protein Expression by Pichia pastoris in DO-Stat Fed-Batch Fermentation"Enzyme and Microbial Technol.. (in press). (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] 熊田 陽一: "Liposome Immunoblotting Assay Utilizing Enhanced Chemiluminescent Reaction Improved by Surfactant"J.Chem.Eng.Japan. (in press). (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Yoichi Kumada, Masumi Maehara, Kanji Tomioka, and Shigeo Katoh: "Liposome Immunoblotting Assay Using a Substrate Forming Precipitate inside Immunoliposomes"Biotechnol. Bioeng.. Vol. 80. 414-418 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Tomoyo Kurokawa, Chun Yeon Lee, Naofumi Shiomi, Atsushi Nakano, and Shigeo Katoh: "Secretion of α-Amylase from Saccharomyces cerevisiae and Pichia pastoris and Characterization of Its C-Terminus with an Anti-Peptide Antibody"J. Chem. Eng. Japan. Vol. 35. 1277-1281 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Chun Yeon Lee, Seok Jea Lee, Kyung Hwan Jung, Shigeo Katoh, and Eun Kyu Lee: "High Dissolved Oxygen Tension Enhances Heterologous Protein Expression by Recombinant Pichia pastoris"Process Biochem.. (in press). (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Chun Yeon Lee, Atsushi Nakano, Naofumi Shiomi, Eun Kyu Lee, and Shigeo Katoh: "Effects of Substrate Feed Rates on Heterologous Protein Expression by Pichia pastoris in DO-Stat Fed-Batch Fermentation"Enzyme and Microbial Technol.. (in press). (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Yoichi Kumada, Masumi Maehara, Naoki Minami, and Shigeo Katoh: "Liposome Immunoblotting Assay Utilizing Enhanced Chemiluminescent Reaction Improved by Surfactant"J. Chem. Eng. Japan. (in press). (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] 熊田 陽一: "Liposome Immunoblotting Assay Using a Substrate Forming Precipitate Inside Immunoliposomes"Biotechnology & Bioengineering. 80巻4号. 414-418 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 黒川 朋世: "Secretion of α-Amylase from Saccharomyces cerevisiae and Pichia pastoris and Characterization of Its C-Terminus with an Anti-Peptide Antibody"Journal of Chemical Engineering of Japan. 35巻12号. 1277-1281 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 加藤 滋雄 他: "Liposome Immunoblotting Assay Using a Substrate Forming Precipitate Inside Immnoliposomes"Biotechnology & Bioengineering. (掲載予定). (2002)

    • Related Report
      2001 Annual Research Report

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Published: 2001-04-01   Modified: 2016-04-21  

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