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Development of a technique for selective chemical labeling and modification of intracellular proteins

Research Project

Project/Area Number 23685038
Research Category

Grant-in-Aid for Young Scientists (A)

Allocation TypeSingle-year Grants
Research Field Chemistry related to living body
Research InstitutionNagaoka University of Technology

Principal Investigator

TSUKIJI Shinya  長岡技術科学大学, 産学融合トップランナーセンター, 特任准教授 (40359659)

Research Collaborator HAMACHI Itaru  京都大学, 大学院工学研究科合成生物化学 専攻, 教授 (90202259)
TAMURA Tomonori  京都大学, 大学院工学研究科合成生物化学 専攻, 大学院生
ISHIDA Manabu  長岡技術科学大学, 産学融合トップランナー養成センター, 博士研究員
KIKUCHI Tamaki  長岡技術科学大学, 生物系, 大学院生
Project Period (FY) 2011-11-18 – 2014-03-31
Project Status Completed (Fiscal Year 2013)
Budget Amount *help
¥15,340,000 (Direct Cost: ¥11,800,000、Indirect Cost: ¥3,540,000)
Fiscal Year 2013: ¥7,670,000 (Direct Cost: ¥5,900,000、Indirect Cost: ¥1,770,000)
Fiscal Year 2012: ¥7,670,000 (Direct Cost: ¥5,900,000、Indirect Cost: ¥1,770,000)
Keywords蛋白質ラベリング / アフィニティラベリング / 蛍光イメージング / 部位特異的変異法 / 化学修飾 / アフィニティラベル化 / 蛋白質 / リガンド / トシル化学 / 細胞 / FKBP12 / 光クロスリンク / 蛋白質-蛋白質相互作用
Research Abstract

Ligand-directed tosyl (LDT) chemistry is a technique that allows selective chemical labeling of specific (native) proteins in cells and in vivo. However, the LDT chemistry is still at the beginning stage, and thus its full potential has yet to be exploited. In this work, we first attempted to apply the LDT chemistry as a novel tool for live-cell fluorescence imaging of endogenous proteins. Using a newly-designed LDT reagent, we were able to selectively label endogenous FKBP12 with an organic fluorescent dye, Oregon Green (OG), in living mammalian cells. It was also possible to visualize the rapamycin-mediated complexation of the OG-labeled FKBP12 and DsRed-fused FRB inside of the cells by FRET imaging. Furthermore, in this work, we demonstrated that the chemical labeling rate and efficiency could be significantly improved by combining the LDT chemistry with the site-directed mutagenesis technique.

Report

(4 results)
  • 2013 Annual Research Report   Final Research Report ( PDF )
  • 2012 Annual Research Report
  • 2011 Annual Research Report
  • Research Products

    (15 results)

All 2013 2012 2011

All Journal Article (5 results) (of which Peer Reviewed: 5 results) Presentation (10 results)

  • [Journal Article] Fluorophore labeling of native FKBP12 by ligand-directed tosyl chemistry allows detection of its molecular interactions in vitro and in living cells2013

    • Author(s)
      T. Tamura, Y. Kioi, T. Miki, S. Tsukiji, I. Hamachi
    • Journal Title

      J. Am. Chem. Soc

      Volume: 135

    • Related Report
      2013 Final Research Report
    • Peer Reviewed
  • [Journal Article] Fluorophore Labeling of Native FKBP12 by Ligand-Directed Tosyl Chemitry Allows Detection of Its Molecular Interaction in Vitro and Living Cells2013

    • Author(s)
      Tomonori Tamura, Yoshiyuki Kioi, Takayuki Miki, Shinya Tsukiji, and Hamachi Itaru
    • Journal Title

      Journal of the American Chemical Society (ACS pubilications)

      Volume: 135 Issue: 18 Pages: 6782-6785

    • DOI

      10.1021/ja401956b

    • Related Report
      2013 Annual Research Report 2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Native FKBP12 engineering by ligand-directed tosyl chemistry : labeling properties and application to photo-cross-linking of protein complexes in vitro and in living cells2012

    • Author(s)
      T. Tamura, S. Tsukiji, I. Hamachi
    • Journal Title

      J. Am. Chem. Soc

      Volume: 134

    • Related Report
      2013 Final Research Report
    • Peer Reviewed
  • [Journal Article] Native FKBP12 engineering by ligand-directed tosyl chemistry : labeling properties and application to photo-cross-linking of protein complexes in vitro and in living cells2012

    • Author(s)
      T.Tamura, S.Tsukiji, I.Hamachi
    • Journal Title

      Journal of the American Chemical Society

      Volume: 134 Issue: 4 Pages: 2216-2226

    • DOI

      10.1021/ja209641t

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] 細胞内在性タンパク質の選択的化学修飾とエンジニアリング2011

    • Author(s)
      築地真也, 石田学, 浜地格
    • Journal Title

      生化学

      Volume: 83

    • NAID

      10029575138

    • Related Report
      2013 Final Research Report 2011 Annual Research Report
    • Peer Reviewed
  • [Presentation] リガンド分子細工による新しいケミカルバイオロジー方法論の開拓2013

    • Author(s)
      築地真也
    • Organizer
      日本化学会第93春季年会
    • Place of Presentation
      立命館大学びわこ・くさつキャンパス,滋賀県 (若い世代の特別講演会)
    • Related Report
      2013 Final Research Report
  • [Presentation] 日本化学会第93春季年会2013

    • Author(s)
      築地真也
    • Organizer
      リガンド分子細工による新しいケミカルバイオロジー方法論の開拓
    • Place of Presentation
      立命館大学びわこ・くさつキャンパス
    • Related Report
      2012 Annual Research Report
  • [Presentation] LDT化学による細胞内FKBP12への光反応基導入と蛋白質間相互作用検出2012

    • Author(s)
      田村朋則, 築地真也, 浜地格
    • Organizer
      第24回生体機能関連化学部会若手の会サマースクール
    • Place of Presentation
      休暇村志賀島,福岡県 (ポスター発表)
    • Related Report
      2013 Final Research Report
  • [Presentation] LDT化学による細胞内蛋白質間相互作用の光クロスリンク検出2012

    • Author(s)
      田村朋則, 築地真也, 浜地格
    • Organizer
      日本ケミカルバイオロジー学会第7回年会
    • Place of Presentation
      京都大学百周年時計台記念館百周年記念ホール,京都府 (ポスター発表)
    • Related Report
      2013 Final Research Report
  • [Presentation] LDT化学による蛋白質工学(1):細胞内蛋白質間相互作用検出への展開2012

    • Author(s)
      田村朋則, 築地真也, 浜地格
    • Organizer
      日本化学会第92春季年会
    • Place of Presentation
      慶應義塾大学日吉・矢上キャンパス,神奈川県 (口頭発表)
    • Related Report
      2013 Final Research Report
  • [Presentation] LDT化学による細胞内蛋白質間相互作用の光クロスリンク検出2012

    • Author(s)
      田村朋則, 築地真也, 浜地格
    • Organizer
      日本ケミカルバイオロジー学会第7回年会
    • Place of Presentation
      京都大学百周年時計台記念館
    • Related Report
      2012 Annual Research Report
  • [Presentation] LDT化学による細胞内FKBP12への光反応基導入と蛋白質間相互作用検出2012

    • Author(s)
      田村朋則, 築地真也, 浜地格
    • Organizer
      第24回生体機能関連化学部会若手の会サマースクール
    • Place of Presentation
      休暇村志賀島
    • Related Report
      2012 Annual Research Report
  • [Presentation] LDT化学による蛋白質工学(1):細胞内蛋白質間相互作用検出への展開2012

    • Author(s)
      田村朋則, 築地真也, 浜地格
    • Organizer
      日本化学会第99春季年会
    • Place of Presentation
      慶應義塾大学日吉キャンパス
    • Related Report
      2011 Annual Research Report
  • [Presentation] LDT化学を用いて光反応基を導入したFKBP12による細胞内蛋白質相互作用検出2011

    • Author(s)
      田村朋則, 築地真也, 浜地格
    • Organizer
      第5回バイオ関連化学シンポジウム
    • Place of Presentation
      つくば国際会議場「エポカルつくば」,茨城県 (ポスター発表)
    • Related Report
      2013 Final Research Report
  • [Presentation] LDT化学を用いて光反応基を導入したFKBP12による細胞内蛋白質相互作用検出2011

    • Author(s)
      田村朋則, 築地真也, 浜地格
    • Organizer
      第5回バイオ関連化学シンポジウム
    • Place of Presentation
      つくば国際会議場「エポカルつくば」
    • Related Report
      2011 Annual Research Report

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Published: 2013-05-15   Modified: 2019-07-29  

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