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Development of ELISA System for Screening of Specific Binding Inhibitors for Src Homology (SH)2 Domain and Phosphotyrosine Interactions

Title
Development of ELISA System for Screening of Specific Binding Inhibitors for Src Homology (SH)2 Domain and Phosphotyrosine Interactions
Authors
Lee S.S.Lee K.I.Yoo J.Jeong M.-J.Park Y.-M.Kwon B.-M.Bae Y.S.Han M.Y.
Ewha Authors
배윤수
SCOPUS Author ID
배윤수scopus
Issue Date
2001
Journal Title
Journal of Biochemistry and Molecular Biology
ISSN
1225-8687JCR Link
Citation
vol. 34, no. 6, pp. 537 - 543
Indexed
SCOPUS WOS scopus
Abstract
In the present study, an in vitro ELISA system to assess the interaction between Src homology (SH)2 domains and phosphotyrosine that contain peptides was established using purified GST-conjugated SH2 proteins and synthetic biotinylated phosphotyrosine that contain oligopeptides. The SH2 domains bound the relevant phosphopeptides that were immobilized in the streptavidin-coated microtiter plate in a highly specific and dose-dependent manner. The epidermal growth factor receptor (EGFR)-, T antigen (T Ag)-, and platelet-derived growth factor receptor (PDGFR)-derived phosphopeptides interacted with the growth factor receptor binding protein (Grb)2/ SH2, Lck/SH2, and phosphatidyl inositol 3-kinase (PI3K) p85/SH2, respectively. No cross-reactions were observed. Competitive inhibition experiments showed that a short phosphopeptide of only four amino acids was long enough to determine the binding specificity. Optimal concentrations of the GST-SH2 fusion protein and phosphopeptide in this new ELISA system for screening the binding blockers were chosen at 2nM and 500nM, respectively. When two candidate compounds were tested in our ELISA system, they specifically inhibited the Lck/ SH2 and/or p85/SH2 binding to the relevant phosphopeptides. Our results indicate that this ELISA system could be used as an easy screening method for the discovery of specific binding blockers of protein-protein interactions via SH2 domains.
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자연과학대학 > 생명과학전공 > Journal papers
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