pBT, a novel vector for tetracycline-regulated yeast three-hybrid assay.
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 microg/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.
Moriyoshi
Koki
K
Department of Molecular and System Biology, Kyoto University Graduate School of Biostudies, Kyoto, 606-8501, Japan. kmoriyos@phy.med.kyoto-u.ac.jp
D004318:Doxycycline / Q000494:pharmacology*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D020218:Response Elements; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D015534:Trans-Activators / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D015534:Trans-Activators; D004318:Doxycycline