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KMID : 0578319950050030217
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Molecules and Cells
1995 Volume.5 No. 3 p.217 ~ p.223
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A Single-step Purification of the Saccharomuces cerevisiae Motichondiral RNA Polymerase Specificity Factor Overproduced in Escherichia coli
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Lee, eun Ah
Yang, Jae Sub/Jang, Sei Heon
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Abstract
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Mitochondrial RNA polymerase of Saccharomyces cervisiae consists of two different proteins: a catalytic core RNA polymerase of 145 kDa (Rpo41p) and a specificity factor of 43 kDa(MtfIp) required for recognizing promoters of the various genes encoded in the mitochondrial genome. A recombinant Mtflp fusion protein was previously partially purified from E. coli by combination of conventional column chromatographies [Mangus, D. A., Jang, S. H., and Jaehning, J. A. (1994) J, Biol. Chem. 269, 26568-26574] . However, the expression level was low and the purification involved multiple steps. Thus, here we have constructed a new expression plasmid coding for the yeast specificity factor and purified it to >95% purity in n single step. The MTFI gene was subcloned into Novagen´s pET15b, which contains an N-terminal six-histidine tag. The resulting plasmid, pET-MTFI, was overexpressed in E. coli, and the fusion protein in inclusion bodies was solubilized with 6 M urea and purified in one step by Ni^(2+)-nitrilotriacetic acid agarose chromatography. The recombinant specificity factor (44kDa) purified in the presence of urea was refolded by dialysis against buffers containing decreasing concentrations of urea. Four mg of the renatured specificity factor were obtained from one gram of cells. The renatured specificity factor in the presence of yeast core polymerase showed promoter selective activity comparable to that purified from yeast in an in vitro selective transcription assay.
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