title: An SH2 domain-based tyrosine kinase assay using biotin ligase modified with a terbium(III) complex.
abstract: 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 Tb(3+) 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.
authors.affiliation: Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka 820-8502, Japan. sueda@bio.kyutech.ac.jp
authors.forename: Shinji
authors.initials: S
authors.lastname: Sueda
authors.forename: Yuki
authors.initials: Y
authors.lastname: Shinboku
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kusaba
mesh_terms: D001710:Biotin / Q000737:chemistry*; D008025:Ligases / Q000737:chemistry*; D008958:Models, Molecular; D016133:Polymerase Chain Reaction; D016944:Sulfolobus / Q000201:enzymology; D013725:Terbium / Q000737:chemistry*; D018909:src Homology Domains; D019061:src-Family Kinases / Q000032:analysis*
chemical_list: D013725:Terbium; D001710:Biotin; D019061:src-Family Kinases; D008025:Ligases