RABBIT LIVER CYTOCHROME-P-450 2B5 - HIGH-LEVEL EXPRESSION OF THE FULL-LENGTH PROTEIN IN ESCHERICHIA-COLI, PURIFICATION, AND CATALYTIC ACTIVITY

Rabbit liver cytochrome P-450 2B5 (P-450 2B5) was expressed in Escheri chia coli using the D(+)-galactose-inducible expression vector pJL-2, containing the full-length cDNA encoding P-450 2B5. Stimulation by gal actose of protein synthesis in the presence of the heme precursor 5-am inolevulinic acid peaked 72 h after addition of the inducer to yield 1 08 nmol membrane-bound P-450 2B5 per liter of culture medium. The reco mbinant enzyme was purified to near homogeneity by a two-column proced ure involving chromatography on DE-52 cellulose and hydroxylapatite. T he hemoprotein was isolated mainly in the low-spin iron configuration and exhibited a reduced GO-difference spectrum with a Soret band at 45 1 nm. Second-derivative spectral analysis in the middle-UV region reve aled that type I binding of 4-nitroanisole to ferric P-450 2B5 abolish ed absorption bands ascribable to tyrosine residues within the polypep tide chain. Pseudo-first-order rates of NADPH-driven reduction of the pigment were lower when reconstituted with NADPH-cytochrome P-450 redu ctase than with the mitochondrial adrenodoxin/NADPH-adrenodoxin reduct ase redox couple. The enzyme was catalytically active toward 4-nitroan isole and androstenedione; metabolic rates were enhanced to different extents by the presence of cytochrome b(5). The recombinant hemoprotei n did not catalyze bioactivation of (N-methyl-N-nitrosamino)-1-(3-pyri dyl)-1-butanone, a potent pulmonary carcinogen. The methods described here should facilitate further studies on the biophysical basis of the complex interactions of P-450 2B5 with its redox partners.