PBT, a novel vector for tetracycline-regulated yeast three-hybrid assay

Abstract

A novel yeast three-hybrid (Y3H) vector pBT was developed, which contains a tetracycline (Tet)-sensitive transactivator (tTA) expression unit and a Tet-responsive element (TRE)-driven 3rd protein expression unit within a single plasmid. To optimize tTA expression levels, several promoters for driving tTA expression were tested, and the weakest human cytomegalovirus (CMV) promoter showed the best induction/background ratio. Culturing yeast cells in different doses of doxycycline (Dox) resulted in a dose-dependent reduction of 3rd protein expression. Screening a cDNA library with pBT successfully identified functional Y3H interactions that could be easily discriminated from Y2H interactions by culturing on Dox-containing plates. At 5.0 μg/ml Dox, Y3H interactions were undetectable by the colony-forming assay under high-stringency selection conditions or by a lacZ colorimetric assay. A low-copy-number version of the pBT vector, pBT(L), completely eliminated the leakage activity of pBT found under low-stringency condition. In conclusion, the pBT system is a useful tool for studying the structures of higher-order protein complexes.