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CONSTRUCTION OF IN VITRO EVOLUTION SYSTEM OF PROTEINS

Research Project

Project/Area Number 09650872
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field 生物・生体工学
Research InstitutionNagoya University

Principal Investigator

NAKANO Hideo  Sch.Agricultral sciences, Nagoya University Assoc.Prof., 農学部, 助教授 (00237348)

Project Period (FY) 1997 – 1998
Project Status Completed (Fiscal Year 1998)
Budget Amount *help
¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1998: ¥1,100,000 (Direct Cost: ¥1,100,000)
Keywordssingle molecule / PCR / cell-free system / transcription / translation / protein synthesis / molecular evolution / library / 1分子 / Polymerase Chain Reaction / 無細胞蛋白合成反応 / 蛋白質ライブラリー
Research Abstract

A novel in vitro method for the generation of a protein library has been developed using the polymerase chain reaction (PCR) amplification of a single DNA molecule followed by in vitro coupled transcription/translation. DNA template encoding green fluorescent protein (GFP) of a jellyfish Aequorea victoria was extensively diluted to one molecule per tube, and then amplified by a total of 80 cycles of PCR with nested primers. The exact number of origins in the amplified DNA fragment was then estimated by directly sequencing a part of the fragment, at which an individual template molecule was marked by PCR with a primer containing three randomized bases. Since the sequehces obtained in 91 independent amplifications were diversified statistically, each amplified fragment was likely originated from a single DNA molecule. In addition, the amplified fragments served as a template for in vitro coupled transcription/translation using T7 RNA polymerase and E.ccli S30 extract. These results suggest that the library obtained by the PCR amplification of a single DNA molecule diluted from a variety of DNA pools is potentially useful in high-throughput generation of protein libraries.

Report

(3 results)
  • 1998 Annual Research Report   Final Research Report Summary
  • 1997 Annual Research Report
  • Research Products

    (9 results)

All Other

All Publications (9 results)

  • [Publications] Shoji Ohuchi et al,: "In vitro method for the generation of protein libraries using PCR amplification of a single DNA molecule and coupled transcription/translation" Nucleic Acids Research. 26. 4339-4346 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Hideo Nakano et al,: "Efficient coupled transcription/translation from PCR template by a hollow fiber membrane bioreactor." Biotechnology & Bioengineering. 印刷中.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Hideo Nakano et al,: "Cell-free protein synthesis systems." Biotechnology Advances. 16. 367-384 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Ohuchi, S., Nakano, H.and Yamane, T.: "In vitro method for the generation of protein libraries using PCR amplification of a single DNA molecule and coupled transcription/translation" Nucleic Acids Research. 26. 4339-4346 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Hideo Nakano, Tomoya Shinbata, Reiko Okumura, Satoshi Sekiguchi, Masatoshi Fujishiro, and Tsuneo Yamane.: "Efficient coupled transcription/translation from PCR template by a hollow fiber membrane bioreactor." Biotechnology & Bioengineering. (in press.).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Hideo Nakano and Tsunoe Yamane.: "Cell-free protein synthesis systems." Biotechnology Advances.16. 367-384 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Shoji Ohuchi,: "In vitro method for the generation of protein libraries using PCR amplification of a single DNA molecule and coupled transcription/translation" Nucleic Acids Research. 26. 4339-4346 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Hideo Nakano, et al.: "Efficient coupled transcription/translation from PCR template by a hollow fiber membrane bioreactor." Biotechnology & Bioengineering. 印刷中.

    • Related Report
      1998 Annual Research Report
  • [Publications] Hideo Nakano: "Cell-free protein synthesis systems." Biotechnology Advances. 16. 367-384 (1998)

    • Related Report
      1998 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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