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

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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 P450a 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 Δ6-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 P450 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.