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 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 to 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 b5. 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.
Lehnerer
M
M
Walther-Straub-Institut für Pharmakologie und Toxikologie, Universität München, Germany.
Schulze
J
J
Petzold
A
A
Bernhardt
R
R
Hlavica
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000072467:Cytochrome P450 Family 2; D003902:Detergents; D004926:Escherichia coli / Q000201:enzymology*; D015870:Gene Expression; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification; D013250:Steroid Hydroxylases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013696:Temperature
D003902:Detergents; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C090279:cytochrome P-450 CYP2B5 (rabbit)