Dissecting the interaction network of multiprotein complexes by pairwise coexpression of subunits in E. coli.
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.
Fribourg
S
S
Institut de Génétique et de Biologie Cellulaire et Moléculaire, CNRS/INSERM/ULP, Collège de France, BP 163, Cedex C.U. de Strasbourg, Illkirch, 67404, France.
Romier
C
C
Werten
S
S
Gangloff
Y G
YG
Poterszman
A
A
Moras
D
D
D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D046911:Macromolecular Substances; D008958:Models, Molecular; D011485:Protein Binding; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D012995:Solubility; D035362:Transcription Factor TFIID; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019473:Transcription Factors, TFII / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D046911:Macromolecular Substances; D021122:Protein Subunits; D011994:Recombinant Proteins; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D051758:Transcription Factor TFIIH