Expression of modified human cytochrome P450 1A2 in Escherichia coli: stabilization, purification, spectral characterization, and catalytic activities of the enzyme.
A full-length human cytochrome P450 (P450) 1A2 cDNA clone and four derivatives in which the 5'-terminus was modified were inserted into the pCW vector and used to transform Escherichia coli cells. Low levels of expression were seen with most of the constructs but high expression levels (245 nmol membrane-bound P450 recovered per liter culture) were achieved when the N-terminus was MALLLAVFL, as reported earlier by Fisher et al. (C. W. Fisher, D. L. Caudle, C. Martin-Wixtrom, L. C. Quattrochi, R. H. Tukey, M. R. Waterman, and R. W. Estabrook, 1992, FASEB J. 6, 759-764). The expressed human P450 1A2 in bacterial membranes was rapidly denatured to cytochrome P420 in the presence of detergents. This denaturation was blocked by the inhibitory ligand alpha-naphthoflavone (alpha NF, 7,8-benzoflavone). Human P450 1A2 was solubilized using high concentrations of sodium cholate and Triton N-101 and could be purified to near homogeneity in high yield in two steps. alpha NF was included in the buffer in the first step and then removed in the second chromatography step along with the detergent. The purified human P450 1A2 was found to be almost completely in the high spin iron configuration, in contrast to P450 1A2 enzymes isolated from rats and rabbits. The enzyme was catalytically active toward the known substrates 7-ethoxyresorufin and phenacetin. The N-terminal appears to be blocked, as is the case for other P450s we have expressed that contain the sequence MALLLAVFL in E. coli. Previously this human P450 has only been available in limited amounts; the methods presented here should facilitate further biochemical and practical studies on this interesting enzyme.
Sandhu
P
P
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Guo
Z
Z
Baba
T
T
Martin
M V
MV
Tukey
R H
RH
Guengerich
F P
FP
D000595:Amino Acid Sequence; D001483:Base Sequence; D001571:Benzoflavones / Q000494:pharmacology; D002384:Catalysis; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017421:Sequence Analysis; D013053:Spectrophotometry
D001571:Benzoflavones; D011994:Recombinant Proteins; C011512:alpha-naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2