Protocol abstract
Nature Protocols 2, - 1368 - 1386 (2007)
Published online: 24 May 2007 | doi:10.1038/nprot.2007.151
Subject Categories: Genomics and proteomics | Isolation, purification and separation | Microbiology and virology | Biochemistry and protein analysis
Identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries
Raffi Tonikian1,2, Yingnan Zhang3, Charles Boone1,2 & Sachdev S Sidhu3
Abstract
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.
- 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.
- Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario, Canada M5G 1L6.
- Department of Protein Engineering, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, USA.
Correspondence to: Sachdev S Sidhu3 e-mail: sidhu@gene.com
nature-products
A-Z product listing
- (NH4)2SO4 (Invitrogen)
- 0.1 M MgCl2 (Invitrogen)
- 0.2-cm gap electroporation cuvet(BTX)
- 0.5 M Tris(Invitrogen)
- 1.0 M H3PO4 (Fisher)
- 1.0 M HEPES(Fisher)
