|
To identify the function of the gene product encoded in the Xenopus laevis cDNA which has high sequence homology with yeast and human TATA-binding protein (TBP) gene [Hashimoto, S., Fujita, H., Hasegawa, S., Roeder, R. G., and Horikoshi, M. (1992) Nucleic Acids Res. 20, 3788], we expressed the putative Xenopus laevis TBP (xTBP) gene by inserting it into a T7 promoter-controlled prokaryotic expression vector, pET-3a. For this purpose, the gene block was amplified by PCR, during which a new NdeI restriction site was introduced at the translation start codon in order to set the reading frame correctly within the expression vector. The resulting plasmid, pET-xTBP, was electrotransformed into E. soli BL21(DE3)pLysS. By IPTG induction, this strain overexpressed a protein of 32 kDa, the size expected of xTBP. From the cell culture induced over a period of 3 h, recombinant xTBP was purified to homogeneity by sequential use of DEAE-Sephacel column chromatography, ammonium sulfate precipitation, heparin-agarose and CM-Sepharose CL-6B column chromatography and finally high performance liquid chromatography with TSK-G3000SW. Bacterially expressed xTBP retained its functional activity in that it can bind to the TATA box of adenovirus type 2 major late promoter (AdMLP) and reconstitute in vitro transcription of the Xenopus laevis tRNAI´3´ gene in Xenopus oocyte 5150 extract.
|