Fluorescence Resonance Energy Transfer-Based Assay for DNA-Binding Protein Tagged by Green Fluorescent Protein

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著者

    • IDEUCHI Hideki
    • Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido
    • TSUCHIDA Shirou
    • Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido
    • WATABE Hiroyuki
    • Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido

抄録

Specific interaction between green fluorescent protein (GFP)-tagged human α- or γ-enolase<SUP>97-242</SUP> (α or γENO<SUP>97-242</SUP>) and the rhodamine-labeled DNA fragment containing the c-<I>myc</I> P2 promoter was detected by a fluorescence resonance energy transfer (FRET)-based assay, designated as a "real-time FRET assay." The approach of donor (GFP) and acceptor (rhodamine) was caused by the association between ENO<SUP>97-242</SUP> and the c-<I>myc</I> P2 promoter, and the time-dependent increase in fluorescence intensity of the reaction mixture was observed at ex=400 nm and em=590 nm. The relative affinity (R<SUB>as</SUB>) of ENO<SUP>97-242</SUP> mutants to the wild type was investigated with a real-time FRET assay, and it was clarified that the amino acids that participated in the interaction existed comparatively broadly. Although it was difficult to measure the absolute value of the affinity for the binding protein by using this method, it was possible to investigate the relative affinity of mutants for the wild type. A real-time FRET assay using the GFP-tagged protein could be used as not only a qualitative, but also as a quantitative analysis, this being the best for investigating the key amino acids in binding proteins.

収録刊行物

  • Bioscience, biotechnology, and biochemistry  

    Bioscience, biotechnology, and biochemistry 70(8), 1921-1927, 2006-08-23 

    Japan Society for Bioscience, Biotechnology, and Agrochemistry

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各種コード

  • NII論文ID(NAID)
    10018528208
  • NII書誌ID(NCID)
    AA10824164
  • 本文言語コード
    ENG
  • 資料種別
    ART
  • ISSN
    09168451
  • NDL 記事登録ID
    8040396
  • NDL 雑誌分類
    ZR7(科学技術--農林水産--農産) // ZR2(科学技術--生物学--生化学) // ZP1(科学技術--化学・化学工業)
  • NDL 請求記号
    Z53-G223
  • データ提供元
    CJP書誌  NDL  J-STAGE 
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