Dissecting the interaction network of multiprotein complexes by pairwise coexpression of subunits in E. coli

J Mol Biol. 2001 Feb 16;306(2):363-73. doi: 10.1006/jmbi.2000.4376.

Abstract

Using the human basal transcription factors TFIID and TFIIH as examples, we show that pairwise coexpression of polypeptides in Escherichia coli can be used as a tool for the identification of specifically interacting subunits within multiprotein complexes. We find that coexpression of appropriate combinations generally leads to an increase in the solubility and stability of the polypeptides involved, which means that large amounts of the resulting complexes can immediately be obtained for subsequent biochemical and structural analysis. Furthermore, we demonstrate that the solubilization and/or the proper folding of a protein by its natural partner can be used as a monitor for deletion mapping to determine precise interaction domains. Coexpression can be used as an alternative or complementary approach to conventional techniques for interaction studies such as yeast two-hybrid analysis, GST pulldown and immunoprecipitation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Escherichia coli / genetics*
  • Gene Expression
  • Genetic Vectors / genetics
  • Humans
  • Macromolecular Substances
  • Models, Molecular
  • Protein Binding
  • Protein Folding
  • Protein Structure, Quaternary
  • Protein Subunits
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Deletion
  • Solubility
  • Transcription Factor TFIID
  • Transcription Factor TFIIH
  • Transcription Factors / chemistry*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Transcription Factors, TFII / chemistry*
  • Transcription Factors, TFII / genetics
  • Transcription Factors, TFII / metabolism*
  • Two-Hybrid System Techniques

Substances

  • Macromolecular Substances
  • Protein Subunits
  • Recombinant Proteins
  • Transcription Factor TFIID
  • Transcription Factors
  • Transcription Factors, TFII
  • Transcription Factor TFIIH