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Complex protein fishing method using proximity dependent reaction of modified transglutaminase

Research Project

Project/Area Number 16K06865
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Research Field Biofunction/Bioprocess
Research InstitutionAkita University

Principal Investigator

Gotoh Takeshi  秋田大学, 理工学研究科, 教授 (10215494)

Research Collaborator Yokota Saki  
Project Period (FY) 2016-04-01 – 2019-03-31
Project Status Completed (Fiscal Year 2018)
Budget Amount *help
¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000)
Fiscal Year 2018: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2017: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2016: ¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000)
Keywordsトランスグルタミナーゼ / プロテオミクス / 組換えタンパク質生産 / タンパク質探索 / FRB / FKBP / バイオテクノロジー / 分子認識 / ナノバイオ
Outline of Final Research Achievements

Two approaches were examined to prepare a fusion protein of transglutaminase (TGase) and bait protein, to determine the possibility that the TGase-bait fusion protein may be used to explore unknown proteins (prey) that bind to the known protein (bait), by proximity-dependent labeling reaction of TGase. First, a modified TGase, in which a TGase-reactive peptide was attached to the C-terminus through a His tag, was prepared and used to couple with a bait protein by its own catalytic activity. However, this approach did not work well probably due to very low concentrations of the proteins. Then, a recombinant E. coli, which was engineered to secrete separately a TGase-FRB fusion and its pro peptide to the periplasm, was constructed. As a result, the TGase fusion protein with FRB attached to the C-terminus could be produced directly in E. coli periplasm and showed an enough TGase activity.

Academic Significance and Societal Importance of the Research Achievements

細胞や組織に発現しているタンパク質の動態を把握し,それらのタンパク質が織りなす相互作用の実態を解析してこれを系統的・包括的に捉えようとするプロテオミクス研究の重要性が近年高かまっている。本研究では,TGaseを利用した近接依存標識反応によって未知タンパク質を標識・同定するために必要な,未知タンパク質と相互作用する既知タンパク質とTGaseの融合体を調製する方法について,有益な方策を提案した。

Report

(4 results)
  • 2018 Annual Research Report   Final Research Report ( PDF )
  • 2017 Research-status Report
  • 2016 Research-status Report
  • Research Products

    (10 results)

All 2019 2018 2017 2016

All Journal Article (3 results) (of which Int'l Joint Research: 1 results,  Peer Reviewed: 1 results) Presentation (7 results) (of which Int'l Joint Research: 2 results)

  • [Journal Article] Effects of rubber elongation factor and small rubber particle protein from rubber producing plants on lipid metabolism in Saccharomyces cerevisiae2019

    • Author(s)
      Yokota Saki, Gotoh Takeshi
    • Journal Title

      Journal of Bioscience and Bioengineering

      Volume: 印刷中

    • NAID

      40022069870

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Biomimetic and Bioinspired Biotechnology2018

    • Author(s)
      Bentley William E., Martin Donald K., Gotoh Takeshi
    • Journal Title

      Biotechnology Journal

      Volume: 13 Issue: 12 Pages: 1800670-1800670

    • DOI

      10.1002/biot.201800670

    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research
  • [Journal Article] Aggregation properties as potential markers for classifying rubber particle proteins2018

    • Author(s)
      Yokota Saki, Gotoh Takeshi
    • Journal Title

      Atlas of Science

      Volume: -

    • Related Report
      2018 Annual Research Report
  • [Presentation] 大腸菌による活性型トランスグルタミナーゼ融合タンパク質の直接生産2019

    • Author(s)
      大池健太,荒木菜那,向山遥香,横田早希,後藤猛
    • Organizer
      化学工学会第84年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] Development of Imprinted L-Dopa-Peptide Silica Particles for Selective Recovery of Ni(II) Ions from Aqueous Solutions2019

    • Author(s)
      Stevens Morlu G.F., Ishibashi Naoya, Adachi Masataka, Yokota Saki, Gotoh Takeshi
    • Organizer
      化学工学会第84年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] Metal Imprinted L-DOPA Peptide Silica Particles for the Selective Recovery of Metals2019

    • Author(s)
      Stevens Morlu G.F., Ishibashi Naoya, Adachi Masataka, Yokota Saki, Gotoh Takeshi
    • Organizer
      秋田化学技術協会第53回研究技術発表会
    • Related Report
      2018 Annual Research Report
  • [Presentation] 改良型トランスグルタミナーゼの生産と自己架橋反応による融合タンパク質の調製2017

    • Author(s)
      佐藤大地,横田早希,佐々木瞬,齋藤 樹,後藤 猛
    • Organizer
      秋田化学技術協会講演会
    • Place of Presentation
      秋田市
    • Year and Date
      2017-03-02
    • Related Report
      2016 Research-status Report
    • Int'l Joint Research
  • [Presentation] Secretory production of self cross-linkable transglutaminase by Brevibacillus2017

    • Author(s)
      大池 健太,横田 早希,佐藤 大地,佐々木 瞬,齋藤 樹,後藤 猛
    • Organizer
      化学系学協会東北大会
    • Related Report
      2017 Research-status Report
  • [Presentation] Cloning and Comparison of Aggregation Properties of HbREF Like Proteins from Ficus carica2017

    • Author(s)
      S. YOKOTA, Y. SUZUKI, K. SAITOH, S. KITAJIMA, N. OHYA, T. GOTOH
    • Organizer
      The 8th International Conference on Materials Engineering for Resources
    • Related Report
      2017 Research-status Report
    • Int'l Joint Research
  • [Presentation] Exploration of a Proximal Protein to Baculovirus GP64 in Sf9 Insect Cells2016

    • Author(s)
      Saki Yokota, Takeshi Gotoh
    • Organizer
      International BioFest 2016
    • Place of Presentation
      メルボルン,オーストラリア
    • Year and Date
      2016-10-24
    • Related Report
      2016 Research-status Report

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Published: 2016-04-21   Modified: 2020-03-30  

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