Expression of rat liver vitamin D3 25‐hydroxylase cDNA in Saccharomyces cerevisiae

Abstract

The cDNA coding for the precursor protein of rat liver mitochondrial vitamin D₃ 25-hydroxylase, cytochrome P450_LMT25, was expressed under the control of the yeast alcohol dehydrogenase I promoter and terminator in Saccharomyces cerevisiae AH22 cells. The transformed years cells produced a P450_LMT25 protein with an almost similar apparent molecular weight as compared with that of the authentic mature enzyme. The expression level of the P450_LMT25 hemoprotein was about 5 × 10⁴ molecules per cell as determined by reduced CO-difference spectra. The mitochondrial fraction prepared from the transformed yeast cells exhibited both 25-hydroxylase activity toward 1α-hydroxyvitamin D₃ and 27-hydroxylase activity toward 5β-cholestane-3α, 7α, 12α-triol in a reconstituted system containing bovine adrenodoxin and NADPH-adrenodoxin reductase.