Identification of 3BP-1 in cDNA expression library by SH3 domain screening

Abstract

This chapter describes a screening procedure to assay for protein–protein interactions using a biotinylated glutathione S-transferase (GST) fusion protein probe to screen cDNA expression libraries. This method is developed to allow nonradioactive probing, which has the additional advantages of a very low background signal and a low incidence of false positives. Because neither the fusion protein probe nor the protein in the expression library is denatured, this assay allows the structural components of the binding reaction to remain intact. This screening procedure offers a practical and highly efficient method of identifying protein–protein interactions. Using this procedure to detect proteins that bound to the Abl SH3 domain, five cDNA clones were isolated out of 7 million cDNA containing plaques that were screened. Of these five, three contained the identical cDNA clone termed "3BP-1," while the other two were identical for a different clone termed "3BP-2." On sequencing these clones, only a short stretch of approximately 40 amino acids was found where sequence similarity existed between these proteins.

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