BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1331-1337 EXPRESSION OF RABBIT CYTOCHROME P-450llE2 IN YEAST AND STABILIZATION OF THE ENZYME BY 4-METHYLPYRAZOLE
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چکیده
A rabbit cytochrome P-450llE2 full-length cDNA was cloned into a yeast episomal plasmid (YEp13) between the copper-responsive yeast metallothionein gene promoter (CUP7) and the iso-I-cytochrome c gene terminator (CYCI), and the cytochrome P-450 was expressed in Saccharomyces cerevisiae. The microsomal fraction prepared from copper-treated cells exhibited a ferrous carbonyl difference spectrum with an absorption maximum at 451 nm and contained approximately 0.07 nmol of P-450llE2 per mg of protein. The P-450llE2 protein expressed in yeast microsomes was catalytically competent as judged by the NADPH-dependent deethylation of N-nitrosodiethylamine and by the oxidation of butanol. Cholate solubilization and polyethylene glycol fractionation of yeast microsomal P-450llE2 yielded a preparation with a markedly lower specific content than that of intact microsomes, but, when 4-methylpyrazole was included during solubilization, the holoenzyme was completely stabilized.
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