A conditional two-hybrid (C2H) system for the detection of protein-protein interactions that are mediated by post-translational modification.
The original bacterial two-hybrid system is widely used but does not permit the study of interactions regulated by PTMs. Here, we have built a conditional two-hybrid (C2H) system, in which bait and prey proteins can be co-expressed in the presence of a modifying enzyme such as a methyltransferase, acetyltransferase, or kinase. Any increase or decrease in interaction due to the modification of the proteins can be measured by an increased or decreased level of reporter gene expression. The C2H system is comprised of eight new vectors based on the Novagen Duet co-expression plasmids. These vectors include two multiple cloning sites per vector as well as a hexahistidine tag or S-tag to aid in purification, if desired. We demonstrate the use of the C2H system to study the dimerization of the yeast protein Npl3, which is increased when methylated by the methyltransferase Hmt1.
Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia. marc.wilkins@unsw.edu.au
Melissa A
MA
Erce
Jason K K
JK
Low
Gene
G
Hart-Smith
Marc R
MR
Wilkins
D008745:Methylation; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011499:Protein Processing, Post-Translational; D011484:Protein-Arginine N-Methyltransferases / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D020798:Two-Hybrid System Techniques
C077734:NPL3 protein, S cerevisiae; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C475182:HMT1 protein, S cerevisiae; D011484:Protein-Arginine N-Methyltransferases