An SH2 Domain-Based Tyrosine Kinase Assay Using Biotin Ligase Modified with a Terbium(III) Complex

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  • SUEDA Shinji
    Department of Bioscience and Bioinformatics, Kyushu Institute of Technology Research Center for Bio-microsensing Technology, Kyushu Institute of Technology
  • SHINBOKU Yuki
    Department of Bioscience and Bioinformatics, Kyushu Institute of Technology
  • KUSABA Takeshi
    Department of Bioscience and Bioinformatics, Kyushu Institute of Technology

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Src homology 2 (SH2) domains are modules of approximately 100 amino acids and are known to bind phosphotyrosine-containing sequences with high affinity and specificity. In the present work, we developed an SH2 domain-based assay for Src tyrosine kinase using a unique biotinylation reaction from archaeon Sulfolobus tokodaii. S. tokodaii biotinylation has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). Here, an SH2 domain from lymphocyte-specific tyrosine kinase was genetically fused to a truncated BCCP, and the resulting fusion protein was labeled through biotinylation with BPL carrying multiple copies of a luminescent Tb3+ complex. The labeled SH2 fusion proteins were employed to detect a phosphorylated peptide immobilized on the surface of the microtiter plate, where the phosphorylated peptide was produced by phosphorylation to the substrate peptide by Src tyrosine kinase. Our assay allows for a reliable determination of the activity of Src kinase lower than 10 pg/μL by a simple procedure.

収録刊行物

  • Analytical Sciences

    Analytical Sciences 29 (5), 491-497, 2013

    社団法人 日本分析化学会

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