Controlled expression of heterologous cytochrome P450e cDNA in Saccharomyces cerevisiae. I. Construction and expression of a complete rat cytochrome P450e cDNA

Abstract

A complete rat cytochrome P450e cDNA was constructed and placed between the yeast-inducible acid phosphatase (PHO5) promoter and terminator. This expression cassette was inserted into the yeast-replicating vector pDP34 which contains the entire yeast 2 μ plasmid element, the dLEU2 and the URA3 marker genes. Saccharomyces cerevisiae was used as a recipient for this construction and was grown under phosphate limitation (induced conditions) in shake flask cultures. Cytochrome P450e production was measured by CO-difference spectra. Cytochrome P450e-specific transcripts were detected by Northern blot analysis and cytochrome P450e proteins by immunoblotting with monoclonal cytochrome P450b antibodies. The intact cytochrome P450e proteins were localized in microsomes. In S. cerevisiae, the cytochrome P450e proteins have been shown to be functional and coupled with yeast NADPH-P450 reductase to form a monooxygenase system.