A tetracycline-regulated expression system is combined with the FLAG- epitope tagging method for conditional expression of potentially toxic proteins in mammalian cells. This strategy allows a controlled expression of exogenous gene products and also provides a unique way of purification. Two- mammalian expression plasmids containing the FLAG sequence and flanking multiple cloning were created for conditional protein expression. The cDNAs encoding human basal transcription factors TBP, TAF(II)155 and the p62 subunit of TFIIH were individually cloned into these vectors and introduced into a HeLa-derived cell line that constitutively expresses a tetracycline- regulated transactivator (tTA). The established clonal human cell lines express FLAG-tagged basal transcription factors in a manner modulated by the amount of tetracycline in the growth medium. In the absence of tetracycline, iTA binds to the DNA recognition sites of the expression plasmid and induces the expression of tagged proteins. When tetracycline is added back to the growth medium, the induced protein start to decay. This provides us with an estimation of the vivo half-lives of TBP and TAF(II)155, which were assessed to be less than 20 and 6 hours, respectively, in HeLa cells. The level of induced proteins in the absence of tetracycline could be further enhanced by including the antibiotic G418 to presumably boost the production of iTA which in turn activates the expression of tagged proteins.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)