Identification of Phosphopeptide Ligands for the Src‐Homology 2 (SH2) Domain of Grb2 by Phage Display

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Abstract
We report here on the identification of phosphopeptide ligands which interact with the Src‐homology 2 (SH2) domain of the adapter protein Grb2 by screening a random peptide library established on phage. Phage were phosphorylated in vitro at an invariant tyrosine residue by a mixture of phosphotyrosine kinases c‐Src, Blk and Syk. Selection of binding motifs was carried out by interaction of the library with the recombinant SH2 domain of Grb2 expressed as a glutathione S‐transferase (GST) fusion protein. Several subsequent cycles of selection led to the enrichment of phage which bound to the GST‐Grb2 SH2 domain only when previously phosphorylated. Sequence analysis revealed that all of the selected phage displayed peptides with the consensus motif Y*M/ENW (Y* denotes phosphotyrosine). One of these peptides, bearing the Y*ENW motif, bound the Grb2 SH2 domain with a threefold higher affinity than the peptide motif Y*VNV derived from the natural ligand She. Thus, phage display can be employed to rapidly identify high affinity ligands to SH2 domains.