Expression of rat liver vitamin D 3 25-hydroxylase cDNA in Saccharomyces cerevisiae

• Published in: FEBS letters Vol. 280; no. 2; pp. 367 - 370
• Main Authors: Akiyoshi-Shibata, Megumi, Usui, Emiko, Sakaki, Toshiyuki, Yabusaki, Yoshiyasu, Noshiro, Mitsuhidel, Okuda, Kyuichiro, Ohkawa, Hideo
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 25.03.1991
• Subjects: 5β-Cholestane-3α, 7α, 12α-triol 27-hydroxylase; Cytochrome P450; Heterologous expression in yeast; Vitamin D 3 25-hydroxylase; Heterologous expression in yeast; 5β-Cholestane-3α, 7α, 12α-triol 27-hydroxylase; Vitamin D 3 25-hydroxylase; Cytochrome P450
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(91)80333-X

The cDNA coding for the precursor protein of rat liver mitochondrial vitamin D 3 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 × 10 4 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 D 3 and 27-hydroxylase activity toward 5β-cholestane-3α, 7α, 12α-triol in a reconstituted system containing bovine adrenodoxin and NADPH-adrenodoxin reductase.