Expression of cytochrome P450 2D6 in Escherichia coli, purification, and spectral and catalytic characterization.
Cytochrome P450 (P450) 2D6 is the classic human liver debrisoquine 4-hydroxylase, the first human P450 for which genetic polymorphism was clearly demonstrated. We prepared 11 different constructs of P450 2D6, with modification at the N-terminus, for expression in Escherichia coli with the vector pCW. These varied considerably in levels of expression of apo- and holoprotein, with the best yield being obtained in a system in which much of the N-terminal hydrophobic segment was removed. Production of holoprotein was highly dependent upon the addition of delta-aminolevulinic acid and FeCl3 to cultures, even though heme production should not be limiting in this system. The expressed protein was not tightly bound to the "heavier" membrane fraction but did not appear to behave as a soluble protein either. A purification strategy was developed involving fractional centrifugation, Triton X-114 phase separation, and flavodoxin affinity chromatography, which led to recovery of apparently electrophoretically homogeneous protein in good yield. Purified P450 2D6 had the expected N-terminal amino acid sequence and catalytic activities toward debrisoquine (4-hydroxylation) and bufuralol (1'-hydroxylation). The availability of a ready source of the recombinant protein should facilitate physical as well as functional studies and antibody production for other uses.
Gillam
E M
EM
Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Australia.
Guo
Z
Z
Martin
M V
MV
Jenkins
C M
CM
Guengerich
F P
FP
D000595:Amino Acid Sequence; D001483:Base Sequence; D019389:Cytochrome P-450 CYP2D6; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006801:Humans; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019389:Cytochrome P-450 CYP2D6