Interaction trap/two-hybrid system to identify loss-of-interaction mutant proteins

Curr Protoc Mol Biol. 2004 Feb;Chapter 20:Unit 20.8. doi: 10.1002/0471142727.mb2008s65.

Abstract

Protein-protein interactions play a critical role in biology. Disrupting a specific protein-protein interaction and studying the resulting phenotype can help elucidate the function of the interaction. A method to rapidly make and identify mutant proteins that no longer bind to a specific partner protein is described. The method takes advantage of the ease of the interaction trap/two-hybrid system and PCR mutagenesis. PCR fusion of the target protein to GFP is used to ensure the protein's open reading frame is not disrupted by mutagenesis. The resulting noninteracting mutant proteins usually result from a single missense mutation, which can easily be identified.

MeSH terms

  • Cloning, Molecular / methods
  • DNA, Complementary / genetics
  • Escherichia coli
  • Gene Library
  • Green Fluorescent Proteins / analysis
  • Green Fluorescent Proteins / genetics
  • Mutation, Missense
  • Open Reading Frames / genetics
  • Plasmids / genetics
  • Point Mutation
  • Polymerase Chain Reaction / methods
  • Protein Interaction Domains and Motifs / genetics
  • Proteins / genetics*
  • Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Two-Hybrid System Techniques*

Substances

  • DNA, Complementary
  • Proteins
  • Saccharomyces cerevisiae Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins