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 interaction of an SH2 domain with a phosphopeptide ligand using scintillation 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 purified from freeze-thaw lysates in high yield by affinity chromatography using immobilized phosphopeptides. For binding assays the phosphopeptide ligands were synthesized with a biotin tag and the FKBP fusion proteins were noncovalently radiolabeled with commercially available [3H]dihydroFK506. Complexes of tritiated SH2 fusion protein and biotinyl-phosphopeptide were then captured on streptavidin-coated SPA beads and counted. The modular protocol is an equilibrium technique that does not employ washing steps or specialized radiochemical syntheses required in other binding assays. The utility of the assay has been demonstrated in an examination of the ligand specificity of the SH2 domains of the tyrosine kinases ZAP70, Syk, and Lck. The methodology is potentially generalizable to any receptor-ligand interaction in which one component can be expressed as a fusion partner with FKBP and the other component can be captured on a SPA bead.
Sonatore
L M
LM
Department of Molecular Design and Diversity, Merck Research Laboratories, Rahway, New Jersey, 07065, USA.
Wisniewski
D
D
Frank
L J
LJ
Cameron
P M
PM
Hermes
J D
JD
Marcy
A I
AI
Salowe
S P
SP
D000595:Amino Acid Sequence; D001710:Biotin / Q000737:chemistry; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D008024:Ligands; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry; D012588:Scintillation Counting; D016559:Tacrolimus / Q000737:chemistry; D022021:Tacrolimus Binding Proteins; D014316:Tritium; D014764:Viral Proteins; D018909:src Homology Domains
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D008024:Ligands; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D014316:Tritium; D001710:Biotin; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D022021:Tacrolimus Binding Proteins; D016559:Tacrolimus