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Creation of Ultra Super Fusion Protein for Antibody Binding and Purification

Research Project

Project/Area Number 25630370
Research Category

Grant-in-Aid for Challenging Exploratory Research

Allocation TypeMulti-year Fund
Research Field Biofunction/Bioprocess
Research InstitutionTokyo Institute of Technology

Principal Investigator

UEDA Hiroshi  東京工業大学, 資源化学研究所, 教授 (60232758)

Co-Investigator(Renkei-kenkyūsha) MATSUYAMA Yuki (OHMURO Yuki)  学術振興会RPD/神戸大学, 大学院工学研究科, 特命助教 (30571088)
Project Period (FY) 2013-04-01 – 2015-03-31
Project Status Completed (Fiscal Year 2014)
Budget Amount *help
¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000)
Fiscal Year 2014: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2013: ¥2,990,000 (Direct Cost: ¥2,300,000、Indirect Cost: ¥690,000)
Keywordsantibody / purification / affinity / avidity / immunoassay / 融合タンパク質 / 抗体 / 結合タンパク / 酵素 / リンカーペプチド / 抗体結合タンパク質 / アビディティー / リンカー工学 / 抗体工学 / 免疫測定 / 結合速度 / 解離速度 / 蛍光プローブ
Outline of Final Research Achievements

Two antibody binding domains from Protein A D (PA) and Protein G C1 (PG) were fused with a linker peptide with various lengths (DDAKK)n, n=0,2,4,6, to create a novel antibody binding protein PAxnPG. When their affinity to Fab and Fc of antibody was evaluated with a biosensor based on biolayer interferometry, the highest binding constant was obtained with n=4 for Fab, and n=6 for Fc, probably reflecting their length between two binding sites. Moreover, the obtained binding constants are several tens-fold higher than those with PA/PG alone. The utility of PAxPG as an antibody detection reagent was also suggested by immunoblot analysis using a fusion protein with chimeric human alkaline phosphatase (AP) with higher catalytic activity and stability than the parent APs from intestinal and placental origins.

Report

(3 results)
  • 2014 Annual Research Report   Final Research Report ( PDF )
  • 2013 Research-status Report
  • Research Products

    (22 results)

All 2015 2014 2013 Other

All Journal Article (12 results) (of which Peer Reviewed: 10 results,  Acknowledgement Compliant: 5 results,  Open Access: 3 results) Presentation (7 results) (of which Invited: 2 results) Book (1 results) Remarks (2 results)

  • [Journal Article] Optimal fusion of antibody binding domains resulted in higher affinity and wider specificity2015

    • Author(s)
      J. Dong*, T. Kojima*, H. Ohashi and H. Ueda *equal contributions
    • Journal Title

      J. Biosci. Bioeng.

      Volume: 印刷中 Issue: 5 Pages: 504-509

    • DOI

      10.1016/j.jbiosc.2015.03.014

    • NAID

      110010017114

    • Related Report
      2014 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] A signal-on fluorosensor based on quench-release principle for sensitive detection of antibiotic rapamycin2015

    • Author(s)
      H.J. Jeong, S. Itayama and H. Ueda
    • Journal Title

      Biosensors

      Volume: 5 Issue: 2 Pages: 131-140

    • DOI

      10.3390/bios5020131

    • Related Report
      2014 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Open flower fluoroimmunoassay: a general method to make fluorescent protein-based immunosensor probes2015

    • Author(s)
      C.I. Chung, R. Makino, J. Dong and H. Ueda
    • Journal Title

      Anal. Chem.

      Volume: 87 Issue: 6 Pages: 3513-3519

    • DOI

      10.1021/acs.analchem.5b00088

    • Related Report
      2014 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] イムノアッセイを目的とした融合タンパク質試薬の設計と生産プロセスの最適化2015

    • Author(s)
      K. Hayashi, Y. Tomozoe, K. Nagai, K. Matsuba, M. Mitsumori, Y. Hiraishi, T. Matsumura, H. Ueda and N. Kamiya
    • Journal Title

      化学工学論文集

      Volume: 41 Pages: 38-42

    • Related Report
      2014 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Simultaneous retention of thermostability and specific activity in chimeric human alkaline phosphatases2015

    • Author(s)
      Y. Sasajima, Y. Kohama, M. Kojima-Misaizu, N. Kurokawa, Y. Hara, J. Dong, M. Ihara and H. Ueda
    • Journal Title

      Mol. Biotechnol.

      Volume: 56 Issue: 10 Pages: 953-961

    • DOI

      10.1007/s12033-014-9774-9

    • Related Report
      2014 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] From Fluorescence Polarization to Quenchbody : Recent Progress in Fluorescent Reagentless Biosensors Based on Antibody and Other Binding Proteins2015

    • Author(s)
      H. Ueda and J.H. Dong
    • Journal Title

      Biochim. Biophys. Acta - Proteins and Proteomics

      Volume: 1844 Issue: 11 Pages: 1951-1959

    • DOI

      10.1016/j.bbapap.2014.06.005

    • Related Report
      2014 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] Ultra Q-bodies : quench-based antibody probes that utilize dye-dye interactions with enhanced antigen-dependent fluorescence2014

    • Author(s)
      R. Abe, H.J. Jeong, D. Arakawa, J.H. Dong, H. Ohashi, R. Kaigome, F. Saiki, K. Yamane, H. Takagi and H.Ueda
    • Journal Title

      Scientific Reports

      Volume: 4 Issue: 1 Pages: 4640-4640

    • DOI

      10.1038/srep04640

    • NAID

      120006582319

    • Related Report
      2014 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] 免疫反応を測れる 光る抗体2014

    • Author(s)
      上田 宏
    • Journal Title

      現代化学

      Volume: 518 Pages: 32-36

    • Related Report
      2014 Annual Research Report
  • [Journal Article] Improved protein-protein interaction assay FlimPIA by the entrapment of luciferase conformation2014

    • Author(s)
      Yuki Ohmuro-Matsuyama, Yuko Hara, and Hiroshi Ueda
    • Journal Title

      Analytical Chemistry

      Volume: (印刷中) Issue: 4 Pages: 2013-2018

    • DOI

      10.1021/ac403065v

    • Related Report
      2013 Research-status Report
    • Peer Reviewed
  • [Journal Article] Open-sandwich immunoassay for sensitive and broad-range detection of a shellfish toxin gonyautoxin2013

    • Author(s)
      Y. Hara, J.H. Dong, and H. Ueda
    • Journal Title

      Anal. Chim. Acta.

      Volume: 793 Pages: 107-113

    • DOI

      10.1016/j.aca.2013.07.024

    • Related Report
      2013 Research-status Report
    • Peer Reviewed
  • [Journal Article] Protein-based Open Sandwich Immuno-PCR for Sensitive Detection of Small Biomarkers2013

    • Author(s)
      S. Hasan, J. Dong, Y. Hara, Y. Morizane, F. Shibasaki and H. Ueda
    • Journal Title

      Analytical Sciences

      Volume: 29 Issue: 9 Pages: 871-876

    • DOI

      10.2116/analsci.29.871

    • NAID

      10031196097

    • ISSN
      0910-6340, 1348-2246
    • Related Report
      2013 Research-status Report
    • Peer Reviewed
  • [Journal Article] 新規蛍光免疫測定素子(Q-body)による各種分子の迅速・簡便な検出法2013

    • Author(s)
      阿部 亮二,大橋 広行,上田 宏
    • Journal Title

      実験医学

      Volume: 31 Pages: 2153-2157

    • Related Report
      2013 Research-status Report
  • [Presentation] 複数結合ドメインの最適配置による高性能抗体結合素子の構築2014

    • Author(s)
      児島智樹,大橋広行,阿部亮二,董 金華,上田 宏
    • Organizer
      第14回日本蛋白質科学会年会
    • Place of Presentation
      横浜
    • Year and Date
      2014-06-27
    • Related Report
      2014 Annual Research Report
  • [Presentation] 抗体結合プローブを用いた蛍光免疫センサーの構築2014

    • Author(s)
      董 金華,児島智樹,大橋広行,阿部亮二,上田 宏
    • Organizer
      第14回日本蛋白質科学会年会
    • Place of Presentation
      横浜
    • Year and Date
      2014-06-25
    • Related Report
      2014 Annual Research Report
  • [Presentation] ドメインの最適配置による高性能抗体結合素子の構築2014

    • Author(s)
      児島 智樹, 大橋 広行, 阿部 亮二, 董 金華,上田 宏
    • Organizer
      第14回蛋白質科学会年会
    • Place of Presentation
      横浜
    • Related Report
      2013 Research-status Report
  • [Presentation] 抗原結合により光る蛍光標識抗体断片Quenchbody (Q-body)の開発2013

    • Author(s)
      上田 宏,阿部 亮二,ジョンヒジン,荒川 大,山根 亨介,高木 広明,芳坂 貴弘
    • Organizer
      第13回日本蛋白質科学会年会
    • Place of Presentation
      鳥取
    • Related Report
      2013 Research-status Report
    • Invited
  • [Presentation] 発光酵素の反応分割による迅速高感度な相互作用検出系の開発2013

    • Author(s)
      上田 宏
    • Organizer
      日本生物工学会第65回大会
    • Place of Presentation
      広島国際会議場
    • Related Report
      2013 Research-status Report
    • Invited
  • [Presentation] 複数結合ドメインの最適配置による高性能抗体結合素子の構築2013

    • Author(s)
      児島 智樹, 大橋 広行, 阿部 亮二, ○上田 宏
    • Organizer
      日本生物工学会第65回大会
    • Place of Presentation
      広島国際会議場
    • Related Report
      2013 Research-status Report
  • [Presentation] 複数結合ドメインの最適配置による高性能抗体結合素子の構築2013

    • Author(s)
      児島 智樹, 大橋 広行, 阿部 亮二, 上田 宏
    • Organizer
      第86回日本生化学会大会
    • Place of Presentation
      横浜
    • Related Report
      2013 Research-status Report
  • [Book] 免疫測定法 基礎から先端まで2014

    • Author(s)
      生物化学的測定研究会,小林典裕,上田 宏,三宅司郎,荒川秀俊編
    • Total Pages
      335
    • Publisher
      講談社
    • Related Report
      2014 Annual Research Report
  • [Remarks] 東京工業大学 資源化学研究所 プロセスシステム工学部門 上田研究室

    • URL

      http://www.ueda.res.titech.ac.jp

    • Related Report
      2014 Annual Research Report
  • [Remarks] 研究業績

    • URL

      http://www.ueda.res.titech.ac.jp/pubs/pubs.html

    • Related Report
      2013 Research-status Report

URL: 

Published: 2014-07-25   Modified: 2019-07-29  

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