BINDING-PROPERTIES OF SH3 PEPTIDE LIGANDS IDENTIFIED FROM PHAGE-DISPLAYED RANDOM PEPTIDE LIBRARIES

Combinatorial libraries have yielded high-affinity ligands for SH3 dom ains of a number of different proteins. We have shown that synthetic p eptides containing these SH3 ligand sequences serve as specific probes of SH3 domains. Direct binding of the N-terminal biotinylated peptide ligands was conveniently detected in ELISA, filter-blotting, and dot- blotting experiments with the use of streptavidin-conjugated enzymes. In some cases, detection of peptide-SH3 interactions required that the biotinylated peptides first were preconjugated with streptavidin to f orm a multivalent complex. Interestingly, these nominally tetravalent SH3 peptide ligands cross-react to varying degrees with different SH3 domains. We have used such complexes to screen lambda cDNA expression libraries and have isolated clones that encode both known and novel SH 3-domain-containing proteins. Based on the success of this methodology , we propose a general strategy by which ligands of a modular domain-c ontaining protein can be isolated from random peptide libraries and us ed to screen cDNA expression libraries systematically for novel modula r domain-containing proteins.