Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme.
A full-length human cytochrome P450 (P450) 3A4 cDNA clone and four derivatives in which the N-terminus was modified were inserted into a pCW vector and used to transform Escherichia coli DH5 alpha cells. Little expression was seen with the native sequence; the highest level of expression (range of 40-110 membrane-bound nmol P450 liter-1) was achieved with a construct (NF14) in which residues 3-12 were deleted. In all of the constructs P450 was found primarily in the membranes. The modified P450 3A4 (construct NF14) showed typical P450 hemoprotein spectra. The protein was purified to electrophoretic homogeneity in a five-step procedure [nominally 23 nmol P450 (mg protein)-1]. For most purposes it was found to be more practical to purify the modified P450 3A4 to approximately 70% homogeneity [nominally 15 nmol P450 (mg protein)-1] in a simple two-step process. The modified P450 3A4 (NF14) or P450 3A4 purified from human liver could be mixed with rabbit liver NADPH-P450 reductase to achieve catalytic activities nearly as high as those found in human liver microsomes (on a nmol P450 basis), but the optimal reconstitution conditions included not only a mixture of phosphatidylserine, L-alpha-dilauroyl- and L-alpha-dioleoyl-sn-glycero-3-phosphocholines, cholate, and cytochrome b5 suggested by others but also glutathione during the preincubation. Several other thiols were found not to substitute in this role. Good catalytic activity was seen for nifedipine oxidation, testosterone 6 beta-hydroxylation, and the 8,9-epoxidation and 3 alpha-hydroxylation of aflatoxin B1, reactions previously ascribed to the enzyme. These procedures provide a relatively convenient and reliable means of producing, purifying, and reconstituting a catalytically active and useful derivative of P450 3A4, a human P450 enzyme that has many roles in the oxidation of drugs and other xenobiotic chemicals.
Gillam
E M
EM
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Baba
T
T
Kim
B R
BR
Ohmori
S
S
Guengerich
F P
FP
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D014169:Transformation, Bacterial
D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; D005978:Glutathione