Binding properties of SH3 peptide ligands identified from phage-displayed random peptide libraries |
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Authors: | Noah G Hoffman Andrew B Sparks J Mark Carter Brian K Kay |
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Institution: | (1) Department of Biology, University of North Carolina at Chapel Hill, 27599-3280 Chapel Hill, NC, USA;(2) Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, 27599-3280 Chapel Hill, NC, USA;(3) Cytogen Corporation, 307 College Road East, 08540-5237 Princeton, NJ, USA |
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Abstract: | Summary Combinatorial libraries have yielded high-affinity ligands for SH3 domains of a number of different proteins. We have shown that synthetic peptides 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 form 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 cDNA expression libraries and have isolated clones that encode both known and novel SH3-domain-containing proteins. Based on the success of this methodology, we propose a general strategy by which ligands of a modular domain-containing protein can be isolated from random peptide libraries and used to screen cDNA expression libraries systematically for novel modular domain-containing proteins. |
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Keywords: | SH3 domains Combinatorial peptide libraries cDNA expression libraries |
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