Rabbit liver cytochrome P-450 2B5: high-level expression of the full-length protein in Escherichia coli, purification, and catalytic activity

• Published in: Biochimica et biophysica acta 1995-08, Vol.1245 (1), p.107-115
• Main Authors: Lehnerer, Michael, Schulze, Johannes, Petzold, Anja, Bernhardt, Rita, Hlavica, Peter
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Aryl Hydrocarbon Hydroxylases; Base Sequence; Catalysis; Catalytic activity; Cloning, Molecular; Cytochrome P-450 2B5; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - isolation & purification; Cytochrome P-450 Enzyme System - metabolism; Cytochrome P450 Family 2; Detergents; Escherichia coli - enzymology; Gene Expression; Heterologous expression; Liver - enzymology; Microsome; Molecular Sequence Data; Rabbits; Recombinant Proteins - isolation & purification; Steroid Hydroxylases - chemistry; Steroid Hydroxylases - isolation & purification; Steroid Hydroxylases - metabolism; Temperature
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(95)00075-M

Rabbit liver cytochrome P-450 2B5 ( P-450 2B5) was expressed in Escherichia coli using the D(+)-galactose-inducible expression vector pJL-2, containing the full-length cDNA encoding P-450 2B5. Stimulation by galactose of protein synthesis in the presence of the heme precursor 5-aminolevulinic acid peaked 72 h after addition of the inducer to yield 108 nmol membrane-bound P-450 2b5 per liter of culture medium. The recombinant enzyme was purified to near homogeneity by a two-column procedure involving chromatography on DE-52 cellulose and hydroxylapatite. The hemoprotein was isolated mainly in the low-spin iron configuration and exhibited a reduced CO-difference spectrum with a Soret band at 451 nm. Second-derivative spectral analysis in the middle-UV region revealed that type I binding of 4-nitroanisole to ferric P-450 2B5 abolished absorption bands ascribable to tyrosine residues within the polypeptide chain. Pseudo-first-order rates of NADPH-driven reduction of the pigment were lower when reconstituted with NADPH-cytochrome P-450 reductase than with the mitochondrial adrenodoxin/NADPH-adrenodoxin reductase redox couple. The enzyme was catalytically active toward 4-nitroanisole and androstenedione; metabolic rates were enhanced to different extents by the presence of cytochrome b 5. The recombinant hemoprotein did not catalyze bioactivation of 4-( N-methyl- N-nitrosamino)-1-(3-pyridyl)-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.