cDNA‐directed expression of rat testosterone 7α‐hydroxylase using the modified vaccinia virus, T7‐RNA‐polymerase system and evidence for 6α‐hydroxylation and Δ₆‐testosterone formation

Abstract

The modified vaccinia virus, T7‐RNA‐polymerase cDNA‐expression system was used to express rat cytochrome P ‐450a. Various parameters such as host‐cell type and density, and duration of infection were tested to optimize the level of expression of cytochrome P 450a enzyme activity. Cytochrome P ‐450a expressed from the cDNA sequence was exclusively incorporated into the membrane‐containing portions of the cell lysates, as expected from its normal association in the liver endoplasmic reticulum. The enzyme displayed a carbon‐monoxide‐reduced‐cytochrome‐ P ‐450a difference spectrum with a Soret maximum of 450 nm. Activity measurements revealed that cytochrome P ‐450a produced three metabolites of testosterone; 7α‐hydroxytestosterone and 6α‐hydroxytestosterone and Δ 6 ‐testosterone at a ratio of about 38:1:1. Under the appropriate conditions, the vaccinia‐virus, T7‐RNA‐polymerase system produces high levels of a single form of cytochrome P 450 in cells that are virtually devoid of endogenous cytochrome P ‐450. Analysis of the cytochrome P ‐450 in its natural membrane‐bound state, as opposed to artificial‐lipid reconstitution studies of purified enzymes, allows accurate and confident measurements of substrate specificities.