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High-speed method for selection of synthetic binding proteins and the application for quantitative detection of a target protein at single-molecule resolution

Research Project

Project/Area Number 15H02006
Research Category

Grant-in-Aid for Scientific Research (A)

Allocation TypeSingle-year Grants
Section一般
Research Field Nanobioscience
Research InstitutionNagoya University

Principal Investigator

MURAKAMI HIROSHI  名古屋大学, 工学研究科, 教授 (10361669)

Co-Investigator(Kenkyū-buntansha) 矢島 潤一郎  東京大学, 大学院総合文化研究科, 准教授 (00453499)
Project Period (FY) 2015-04-01 – 2019-03-31
Project Status Completed (Fiscal Year 2018)
Budget Amount *help
¥41,990,000 (Direct Cost: ¥32,300,000、Indirect Cost: ¥9,690,000)
Fiscal Year 2018: ¥8,320,000 (Direct Cost: ¥6,400,000、Indirect Cost: ¥1,920,000)
Fiscal Year 2017: ¥8,320,000 (Direct Cost: ¥6,400,000、Indirect Cost: ¥1,920,000)
Fiscal Year 2016: ¥8,320,000 (Direct Cost: ¥6,400,000、Indirect Cost: ¥1,920,000)
Fiscal Year 2015: ¥17,030,000 (Direct Cost: ¥13,100,000、Indirect Cost: ¥3,930,000)
Keywordsbinding protein / screening / translation / single-molecule / 人工抗体 / 1分子定量 / タンパク質 / 1分子定量 / 一分子定量 / 抗体 / 1分子 / 血管内皮細胞増殖因子受容体
Outline of Final Research Achievements

The purpose of this research was to develop a high-speed method for selection of synthetic binding proteins (SBPs) and quantitative detection of the corresponding target proteins at single-molecule resolution. First, we modified our previous high-speed method of screening functional polypeptides (transcription-translation coupled with association of puromycin linker [TRAP] display; JACS, 2013) for SBPs selection. Then, using the improved TRAP display, we demonstrated successful selection of high-affinity (nM level) SBPs against model target proteins, EGFR1 and HER2. Finally, we applied the modified method to obtain several SBPs against low-copy-number proteins and to detect a target protein immobilized on a glass plate using a fluorescently labeled SBP. The improved version of the TRAP display for SBP selection and single-molecule detection of target proteins with the obtained SBPs could become a basic high-precision technology for protein quantification at single-molecule resolution.

Academic Significance and Societal Importance of the Research Achievements

1細胞におけるタンパク質の分子数は、タンパク質の種類によって大きく異なる。これら様々な数からなるタンパク質群の定量は、生物科学において個々の細胞の個性を理解するための重要な課題である。そこで近年、タンパク質を1分子単位で定量する新手法の開発が進められている。このボトルネックは、標的に対して高い特異性と親和性を持った高性能抗体の作製である。本研究では、簡便に高速で人工抗体を作製する手法を開発した。さらに得られた人工抗体を用いてガラス基板上に固定化した標的を1分子単位で検出することができることを示した。本方法は、タンパク質の1分子定量を可能にする重要な基盤技術となると期待される。

Report

(5 results)
  • 2018 Annual Research Report   Final Research Report ( PDF )
  • 2017 Annual Research Report
  • 2016 Annual Research Report
  • 2015 Annual Research Report
  • Research Products

    (17 results)

All 2019 2018 2017 2016 2015

All Journal Article (4 results) (of which Peer Reviewed: 3 results,  Open Access: 1 results,  Acknowledgement Compliant: 2 results) Presentation (13 results) (of which Int'l Joint Research: 6 results,  Invited: 5 results)

  • [Journal Article] Exploring of minimal RNA substrate of flexizymes2019

    • Author(s)
      Tomoshige Fujino, Taishi Kondo, Hiroaki Suga, and Hiroshi Murakami
    • Journal Title

      ChemBioChem

      Volume: 印刷中

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Circular orientation fluorescence emitter imaging (COFEI) of rotational motion of motor proteins2018

    • Author(s)
      Mitsuhiro Sugawa, Tomoko Masaike, Nagisa Mikami, Shin Yamaguchi, Keitaro Shibata, Kei Saito, Fumihiko Fujii, Yoko Y Toyoshima, Takayuki Nishizaka, Junichiro Yajima.
    • Journal Title

      Biochemical and biophysical research communications

      Volume: 504 Issue: 4 Pages: 709-714

    • DOI

      10.1016/j.bbrc.2018.08.178

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method2017

    • Author(s)
      Taniguchi, N., Murakami, H.
    • Journal Title

      Methods Mol Biol

      Volume: 1498 Pages: 339-347

    • DOI

      10.1007/978-1-4939-6472-7_22

    • ISBN
      9781493964703, 9781493964727
    • Related Report
      2016 Annual Research Report
    • Acknowledgement Compliant
  • [Journal Article] In Vitro Selection Combined with Ribosomal Translation Containing Non-proteinogenic Amino Acids2016

    • Author(s)
      Tomoshige Fujino and Hiroshi Murakami
    • Journal Title

      The Chemical Record

      Volume: 16 Issue: 1 Pages: 365-377

    • DOI

      10.1002/tcr.201500239

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Presentation] CYK4 promotes the flexibility in the sidward stepping by MKLP1 kinesin-62019

    • Author(s)
      Yohei Maruyama, Mitsuhiro Sugawa, Masanori Mishima, Junichiro Yajima
    • Organizer
      Mitotic Spindle symposium. From living and synthetic systems to theory
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Improved TRAP Display for Selection of Monobodies, Nanobodies, and Macrocyclic Peptides2018

    • Author(s)
      近藤 太志,瀬崎 貴大,鬼頭 清太,石崎 敬悟,藤野 公茂,村上 裕
    • Organizer
      第10回国際ペプチドシンポジウム
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Improved TRAP display for selection of binding peptides against multiple target proteins2018

    • Author(s)
      近藤 太志,瀬崎 貴大,鬼頭 清太,石崎 敬悟,藤野 公茂,村上 裕
    • Organizer
      he 17th Akabori Conference(German-Japanese Symposium on Peptide Science)
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research
  • [Presentation] 無細胞翻訳系を用いた 進化分子工学・合成生物学2018

    • Author(s)
      村上裕
    • Organizer
      第11回ChemBioハイブリッドレクチャー
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] TRAP提示法を用いた迅速な人工抗体創製2018

    • Author(s)
      村上裕
    • Organizer
      第49回 中部化学関係学協会支部連合秋季大会
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] TRAP提示法を用いた機能性ペプチド・人工抗体選択2017

    • Author(s)
      村上裕
    • Organizer
      化学工学会第49回秋季大会
    • Related Report
      2017 Annual Research Report
    • Invited
  • [Presentation] 2′-OMeオリゴヌクレオチドを用いたTRAP displayの改良2017

    • Author(s)
      近藤太志、石﨑敬悟、藤野公茂、村上裕
    • Organizer
      日本ケミカルバイオロジー学会第12回年会
    • Related Report
      2017 Annual Research Report
  • [Presentation] 改良TRAP提示法によるmRNAへの人工抗体提示の高効率化2017

    • Author(s)
      近藤太志、石﨑敬悟、藤野公茂、村上裕
    • Organizer
      第11回バイオ関連化学シンポジウム
    • Related Report
      2017 Annual Research Report
  • [Presentation] Improved TRAP display for high-speed selection of antibodies from trillions of candidates2017

    • Author(s)
      近藤太志、石﨑敬悟、藤野公茂、村上裕
    • Organizer
      the 2nd International Symposium on Biofunctional Chemistry (ISBC2017)
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research
  • [Presentation] CIK4 induces the large fluctuations of the left-handed rotational movement of dimeric kinesin62017

    • Author(s)
      丸山洋平、須河光弘、三嶋将紀、矢島潤一郎
    • Organizer
      Biophysical society 62nd annual meeting
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research
  • [Presentation] 翻訳系を用いた進化分子工学と合成生物学2017

    • Author(s)
      村上裕
    • Organizer
      ChemBio講演会2017-1
    • Place of Presentation
      京都大学
    • Related Report
      2016 Annual Research Report
    • Invited
  • [Presentation] High-speed in vitro display method for selection of functional peptides and proteins2015

    • Author(s)
      中山紗由美、藤野公茂、村上裕
    • Organizer
      2015環太平洋国際化学会議
    • Place of Presentation
      コンベンションセンター(ハワイ、ホノルル)
    • Year and Date
      2015-12-15
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research
  • [Presentation] TRAP提示法を用いた高速人工抗体作製法の開発2015

    • Author(s)
      中山紗由美、藤野公茂、村上裕
    • Organizer
      第9回バイオ関連化学シンポジウム
    • Place of Presentation
      熊本大学(熊本県熊本市)
    • Year and Date
      2015-09-10
    • Related Report
      2015 Annual Research Report

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Published: 2015-04-16   Modified: 2020-03-30  

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