Nucleic Acids Research Advance Access published online on December 2, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn969
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Methods paper |
pBT, a novel vector for tetracycline-regulated yeast three-hybrid assay
Department of Molecular and System Biology, Kyoto University Graduate School of Biostudies, Kyoto, 606-8501, Japan
*To whom correspondence should be addressed. Tel: +81 75 753 4407; Fax: +81 75 753 4404; Email: kmoriyos{at}phy.med.kyoto-u.ac.jp
Received September 4, 2008. Revised October 20, 2008. Accepted November 15, 2008.
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.