Expression of human cytochrome p450 3A4 gene in Schizosaccharomyces pombe

Abstract

Heterologous expression systems can be utilized to great advantage in the study of cytochrome P450 enzymes. P450 3A4 is one of the major forms of cytochrome P450 found in liver. It is also involved in the metabolism of numerous widely used drugs and xenobiotics. In the present study human liver cytochrome P450 3A4 gene was transferred into the fission yeast Schizosaccharomyces pombe via two different S. pombe expression vectors carrying thiamine repressible promoter - nmt1 (pREP42) and constitutive promoter - adh1 (pART1). Heterologously expressed cytochrome P450 3A4 was detected in the cells grown in minimal (EMM) or rich medium (YEL) containing 0.5% (w/v) glucose. A typical cytochrome P450 peak for 3A4 was observed at 448 nm in microsomal fraction. The presence of heterologous expression of 3A4 form was also determined by SDS-PAGE and it molecular mass was identified as 52 kDa. The enzyme activity was confirmed by HPLC analysis, using testosterone as substrate.