Characterizing Protein-Protein Interactions Using Deep Sequencing Coupled to Yeast Surface Display

Methods Mol Biol. 2018:1764:101-121. doi: 10.1007/978-1-4939-7759-8_7.

Abstract

In this chapter, we discuss a method to determine the affinity and specificity of nearly all single-point mutants for a full-length protein binder. This method combines deep sequencing, comprehensive mutagenesis, yeast surface display, and fluorescence-activated cell sorting. This approach has been used to study sequence-function relationships for protein-protein interactions. The data can be used to determine the fine conformational epitope on the protein binder.

Keywords: Conformational epitope mapping; Deep sequencing; FACS; Nicking mutagenesis; Yeast surface display.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Binding Sites
  • Cell Adhesion Molecules / chemistry
  • Cell Adhesion Molecules / genetics
  • Cell Adhesion Molecules / metabolism*
  • Cell Separation
  • Epitope Mapping / methods*
  • Epitopes / chemistry*
  • Epitopes / genetics
  • Flow Cytometry
  • High-Throughput Nucleotide Sequencing / methods*
  • Mutagenesis
  • Peptide Library
  • Protein Binding
  • Protein Interaction Domains and Motifs*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • Cell Adhesion Molecules
  • Epitopes
  • Peptide Library
  • Saccharomyces cerevisiae Proteins