Expression of Bovine Cytochrome P450c17 cDNA inSaccharomyces cerevisiae

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Abstract
We constructed expression plasmids for bovine adrenal cytochrome P450c17 (P450c17) by inserting the corresponding cDNA between the yeast alcohol dehydrogenase I promoter and terminator of the expression vector pAAH5. Plasmids pAα1 and pAα2 contained the entire coding region for bovine P450c17, whereas pACα1 included the cDNA coding for chimeric P450cα consisting of the amino-terminal 45 amino acid residues of rat P450c and the carboxy-terminal 482 amino acid residues of bovine P450c17. The transformed Saccharomyces cerevisiae AH22/pAα1, AH22/pAα2, and AH22/pACα1 cells produced about 1 × 105, 1 × 105, and 2 × 104 molecules per cell of the corresponding P450 hemoproteins, respectively. On incubation with the cultures of each of the three strains, progesterone was specifically converted into 17α-hydroxyprogesterone, which was not further converted into androstenedione, indicating that the three strains showed 17α-hydroxylase activity, but almost no C17,20-lyase activity. The microsomal fraction prepared from the AH22/pAα1 cells showed 17α-hydroxylase activity toward progesterone and pregnenolone to higher extents, and exhibited C17,20-lyase activity toward 17α-hydroxypregnenolone to a lesser extent and almost no C17,20-lyase activity toward 17α-hydroxyprogesterone. These results indicated that bovine P450c17 synthesized in S. cerevisiae cells manifests the 17α-hydroxylase activity, but not the C17,20-lyase activity.

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