Escherichia coli expression and characterization of cytochromes P450 2B11, 2B1, and 2B5.
Dog CYP2B11, rat CYP2B1, and rabbit CYP2B5 have been expressed in Escherichia coli from cDNAs modified at the N-terminus (Barnes et al., 1991, Proc. Natl. Acad. Sci. USA 88, 5597-5601). Using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps), solubilized membranes representing > 100 nmol of P450 2B11, > 35 nmol of P450 2B1, and > 7 nmol of P450 2B5 were efficiently extracted (40-70% yield) from a 1-liter culture. Chaps-solubilized preparations produced a reduced CO/reduced difference spectrum devoid of P420 and were used directly in a reconstituted system. The E. coli-expressed 2B enzymes retained the same functional characteristics as the purified hepatic enzymes or enzymes expressed in COS cells in terms of androstenedione metabolite profiles. Hydroxylation rates were determined under a variety of conditions, including two concentrations of NADPH-cytochrome P450 reductase (2 and 16 nmol/nmol P450) and the absence or presence of cytochrome b5 (2 nmol/nmol P450). The androstenedione hydroxylase activities of expressed 2B1 and 2B5 were stimulated by cytochrome b5, whereas P450 2B11 was inhibited slightly by cytochrome b5. Purified expressed 2B11 (specific content, 8 nmol/mg protein) had similar activities as the Chaps-solubilized membrane preparation. The solubilized membranes containing 2B11 were also tested with 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB). Three major metabolites, 2-hydroxy-4,5,2',4',5'-pentachlorobiphenyl, 3-hydroxy-2,4,5,2',4',5'-hexachlorobiphenyl, and 2-hydroxy-3,4,5,2',4',5'-hexachlorobiphenyl were produced from 245-HCB. These metabolites are identical to those produced by 2B11 purified from liver microsomes. The 245-HCB hydroxylation rates were similar for E. coli-expressed 2B11, dog liver microsomes, and purified liver 2B11. When only the second codon in the 2B1 was changed to GCT, > 25 nmol of P450 was extracted from a 1-liter culture, suggesting that the full Barnes et al. modification scheme may not be necessary for high-level expression. An efficient method of expressing, extracting, and analyzing different P450 2B enzymes has thus been achieved. In addition, rabbit P450 2B5, which has never been purified from liver, as well as different P450 2B mutants can now be expressed at much higher levels than previously reported. The ability to express different 2B wild-type and mutant P450s in E. coli provides an excellent opportunity to study the molecular basis of species differences in substrate metabolism.
John
G H
GH
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721.
Hasler
J A
JA
He
Y A
YA
Halpert
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002793:Cholic Acids; D003001:Cloning, Molecular; D019362:Cytochrome P-450 CYP2B1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D000072467:Cytochrome P450 Family 2; D017931:DNA Primers; D003902:Detergents; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism
D002793:Cholic Acids; D017931:DNA Primers; D003902:Detergents; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D019362:Cytochrome P-450 CYP2B1; D000072467:Cytochrome P450 Family 2; C090278:cytochrome P-450 CYP2B11 (dog); C090279:cytochrome P-450 CYP2B5 (rabbit); C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate