Analytical Sciences

Abstract − Analytical Sciences, 29(5), 491 (2013).

An SH2 Domain-Based Tyrosine Kinase Assay Using Biotin Ligase Modified with a Terbium(III) Complex
Shinji SUEDA,*,** Yuki SHINBOKU,* and Takeshi KUSABA*
*Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka 820-8502, Japan
**Research Center for Bio-microsensing Technology, Kyushu Institute of Technology, 1-1 Sensui-cho, Tobata, Kitakyushu 804-8550, Japan
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.