title: Truncated human P450 2D6: expression in Escherichia coli, Ni(2+)-chelate affinity purification, and characterization of solubility and aggregation.
abstract: Cytochrome P450 2D6 is one of the clinically important drug-oxidizing enzymes in human liver. We constructed an Escherichia coli expression vector to obtain large amounts of this microsomal hemoprotein in a form suitable for purification and further structural analysis. The N-terminal 25 amino acids, which presumably serve as a membrane anchor, were replaced by codons for a [His]6 tag to increase solubility and to allow for rapid purification by Ni(2+)-chelate affinity chromatography. P450 2D6 apoprotein was synthesized under practically all growth conditions, whereas formation of heme-containing holoenzyme strictly depended on addition of the heme precursor delta-aminolevulinic acid to the E. coli culture. The truncated P450 was purified from the soluble cytosolic fraction to electrophoretic homogeneity (7 nmol of P450/mg protein) by affinity chromatography on Ni(2+)-nitrilotriacetate-agarose. The purified protein exhibited a CO-reduced difference spectrum with a delta max at 450 nm and no detectable P420. Kinetic analysis revealed a Km value for bufuralol 1'-hydroxylation similar to the Km of the native full-length enzyme purified from human liver microsomes. To characterize the purified truncated protein with respect to hydrodynamic properties, we performed sedimentation velocity and sedimentation equilibrium analysis. These studies demonstrated that approximately 50% of the protein was in a highly aggregated state. Another 30% consisted of a single protein species with an approximated molecular weight of 200,000 and the residual 20% represented at least two other species with lower molecular weights. To prevent formation of such unexpectedly high aggregation states, purification was also performed in the presence of "nonaethyleneglycol monododecyl ether" (C12E9), a nonionic, chemically defined detergent often used in attempts to crystallize membrane proteins. Over 80% of this preparation was found to consist of a single protein species with a M(r) of 62,000 indicating a monomeric state. These data demonstrate that (i) N-terminal truncation of P450 2D6 does not principally interfere with heme incorporation and function of the enzyme, (ii) deletion of the hydrophobic N-terminus leads to a protein product which can be recovered largely from the soluble E. coli fraction, and (iii) this soluble truncated protein is highly aggregated unless detergents are added to maintain it in a monomeric state.
authors.affiliation: Department of Pharmacology, University of Basel, Switzerland.
authors.forename: A C
authors.initials: AC
authors.lastname: Kempf
authors.forename: U M
authors.initials: UM
authors.lastname: Zanger
authors.forename: U A
authors.initials: UA
authors.lastname: Meyer
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002248:Carbon Monoxide; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D019389:Cytochrome P-450 CYP2D6; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli; D006639:Histidine; D006801:Humans; D007502:Iron Chelating Agents; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009532:Nickel; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D016324:Sequence Tagged Sites; D012995:Solubility; D013053:Spectrophotometry
chemical_list: D003062:Codon; D017931:DNA Primers; D007502:Iron Chelating Agents; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D006639:Histidine; D009532:Nickel; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019389:Cytochrome P-450 CYP2D6