Expression of modified human cytochrome P450 2E1 in Escherichia coli, purification, and spectral and catalytic properties.
Human cytochrome P450 (P450) 2E1 is of interest because of its role in the oxidation of numerous drugs and carcinogens. The purification of the protein from human liver is difficult, and we report the development of a system for relatively high-level expression in Escherichia coli. A cDNA was prepared from liver cDNA by polymerase chain reaction methods and several variants with modified 5'-termini were constructed. Analysis of seven of these indicated that the highest levels of expression were found when the first 21 codons of the native sequence were deleted and the Trp immediately following the resulting N-terminal Met was changed to Ala (GCT). Levels of 40-nmol membrane-bound P450 2E1 (liter culture)-1 were routinely recovered. The recombinant P450 2E1 was purified to electrophoretic homogeneity from the bacterial membranes in two ion-exchange steps in > 80% yield. Ferric P450 2E1 was isolated in a mixed spin state. The enzyme was active in chlorzoxazone 6-hydroxylation; the addition of human liver cytochrome b5 lowered the Km for the substrate and increased Vmax. N-Terminal amino acid sequence analysis yielded the expected first 21 residues. The expression system should facilitate the availability of human P450 2E1 and antibodies for studies of the enzyme.
Gillam
E M
EM
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Guo
Z
Z
Guengerich
F P
FP
D001483:Base Sequence; D002384:Catalysis; D002753:Chlorzoxazone / Q000378:metabolism; D003001:Cloning, Molecular; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006900:Hydroxylation; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D013053:Spectrophotometry
D012333:RNA, Messenger; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; D002753:Chlorzoxazone