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

Nature Protocols volume 2, pages 1368–1386 (2007)Cite this article

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.

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Figure 1: A flow diagram of the experimental components required in identifying consensus profiles for PRMs.
Figure 2: Cloning strategy to generate GST-PRM fusions.
Figure 3: Phagemid vectors designed for peptide display.
Figure 4: Mutagenesis for library construction.
Figure 5: Affinity selection process.
Figure 6: Consensus profile identification for the human ERBIN PDZ domain.
Figure 7: In vitro synthesis of heteroduplex CCC-dsDNA.

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Authors and Affiliations

  1. Department of Molecular and Medical Genetics, University of Toronto, #4398 Medical Sciences Building, 1 King's College Circle, University of Toronto, Toronto, M5S 1A8, Ontario, Canada

    Raffi Tonikian & Charles Boone

  2. Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, M5G 1L6, Ontario, Canada

    Raffi Tonikian & Charles Boone

  3. Department of Protein Engineering, Genentech Inc., 1 DNA Way, South San Francisco, 94080, California, USA

    Yingnan Zhang & Sachdev S Sidhu

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  1. Raffi Tonikian
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  4. Sachdev S Sidhu
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Correspondence to Sachdev S Sidhu.

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Tonikian, R., Zhang, Y., Boone, C. et al. Identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries. Nat Protoc 2, 1368–1386 (2007). https://doi.org/10.1038/nprot.2007.151

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