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A conditional two‐hybrid (C2H) system for the detection of protein–protein interactions that are mediated by post‐translational modification
Author(s) -
Erce Melissa A.,
Low Jason K. K.,
HartSmith Gene,
Wilkins Marc R.
Publication year - 2013
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201200477
Subject(s) - methyltransferase , plasmid , cloning (programming) , acetyltransferase , yeast , expression vector , chemistry , biochemistry , gene , computational biology , biology , target protein , recombinant dna , computer science , methylation , acetylation , programming language
The original bacterial two‐hybrid system is widely used but does not permit the study of interactions regulated by PTM s. Here, we have built a conditional two‐hybrid ( C 2 H ) 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 C 2 H system is comprised of eight new vectors based on the N ovagen D uet 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 C 2 H system to study the dimerization of the yeast protein N pl3, which is increased when methylated by the methyltransferase H mt1.