Establishment of stable cell lines expressing potentially toxic proteins by tetracycline-regulated and epitope-tagging methods.
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 protein purification. Two mammalian expression plasmids containing the FLAG sequence and flanking multiple cloning sites were created for conditional protein expression. The cDNAs encoding human basal transcription factors TBP, TAFII55 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, tTA 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 starts to decay. This provides us with an estimation of the in vivo half-lives of TBP and TAFII55, 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 tTA which in turn activates the expression of tagged proteins.
Wu
S Y
SY
Department of Biochemistry, University of Illinois at Urbana-Champaign 61801, USA.
Chiang
C M
CM
D001483:Base Sequence; D002460:Cell Line; D000939:Epitopes; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013752:Tetracycline / Q000494:pharmacology*; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics
D000939:Epitopes; D011994:Recombinant Proteins; D014157:Transcription Factors; D013752:Tetracycline