Identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries

Signaling complexes usually involve multidomain proteins containing catalytic domains and peptide recognition modules (PRMs), which mediate protein-protein interactions and assemble complexes by binding to ligands containing a core sequence motif. Concomitant to large-scale physical interaction screening, considerable effort has been devoted toward the elucidation of consensus profiles for common PRMs. We describe herein a robust and proven protocol to generate consensus profiles for PRMs using phage-displayed peptide libraries. The initial phase of the protocol entails the cloning, expression and purification of PRMs as fusion proteins, in addition to the construction of highly diverse phage-displayed peptide libraries. The affinity selection process described thereafter enables a single researcher to efficiently probe the recognition profiles of numerous PRMs in a 1 week time period.