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
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- SHINBOKU Yuki
- Department of Bioscience and Bioinformatics, Kyushu Institute of Technology
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- 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
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- Analytical Sciences
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Analytical Sciences 29 (5), 491-497, 2013
社団法人 日本分析化学会
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詳細情報 詳細情報について
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- CRID
- 1390001204260769920
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- NII論文ID
- 10031170976
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- NII書誌ID
- AA10500785
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- COI
- 1:STN:280:DC%2BC3snislejsQ%3D%3D
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- ISSN
- 13482246
- 09106340
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- NDL書誌ID
- 024473565
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- PubMed
- 23665620
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- 本文言語コード
- en
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- データソース種別
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- JaLC
- NDL
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- PubMed
- CiNii Articles
- KAKEN
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