THE UTILITY OF FK506-BINDING PROTEIN AS A FUSION PARTNER IN SCINTILLATION PROXIMITY ASSAYS - APPLICATION TO SH2 DOMAINS

Methodology has been developed which gives a specific measure of the i nteraction of an SH2 domain with a phosphopeptide ligand using scintil lation proximity assay (SPA) technology. Recombinant SH2 domains were expressed from a T7 RNA polymerase-based vector in Escherichia coli as fusions to the C-terminus of the FK506-binding protein (FKBP) and pur ified from freeze-thaw lysates in high yield by affinity chromatograph y using immobilized phosphopeptides. For binding assays the phosphopep tide ligands were synthesized with a biotin tag and the FKBP fusion pr oteins were noncovalently radiolabeled with commercially available [H- 3]dihydroFK506. Complexes of tritiated SH2 fusion protein and biotinyl -phosphopeptide were then captured on streptavidin-coated SPA beads an d counted. The modular protocol is an equilibrium technique that does not employ washing steps or specialized radiochemical syntheses requir ed in other binding assays. The utility of the assay has been demonstr ated in an examination of the ligand specificity of the SH2 domains of the tyrosine kinases ZAP70, Syk, and Lck. The methodology is potentia lly generalizable to any receptor-ligand interaction in which one comp onent can be expressed as a fusion partner with FKBP and the other com ponent can be captured on a SPA bead. (C) 1996 Academic Press, Inc.