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.
Tonikian
Raffi
R
Department of Molecular and Medical Genetics, University of Toronto, #4398 Medical Sciences Building, 1 King's College Circle, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Zhang
Yingnan
Y
Boone
Charles
C
Sidhu
Sachdev S
SS
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D019151:Peptide Library; D010455:Peptides / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D004269:DNA, Bacterial; D019151:Peptide Library; D010455:Peptides