Expression of cytochrome P450 2A6 in Escherichia coli: purification, spectral and catalytic characterization, and preparation of polyclonal antibodies.
Cytochrome P450 (CYP) 2A6 is the principal human enzyme catalyzing coumarin 7-hydroxylation and is known to be involved in the metabolism of halothane, nicotine, and metabolic activation of butadiene and nitrosamines. In this paper expression of CYP2A6 in Escherichia coli is reported. In order to achieve expression, the N-terminus of protein was modified by PCR mutagenesis. The N-terminal variant with only a single amino acid change showed expression of 210 nmol of CYP2A6/liter of culture. Recombinant CYP2A6 protein was purified to electrophoretic homogeneity and further characterized. Absolute spectra were typical for CYP proteins and indicated low spin characteristics of isolated protein. Due to a hydrophobic segment the N-terminal amino acid sequence of recombinant CYP2A6 was blocked. The N-terminal formylmethionine block was removed by mild acid treatment. Purified CYP2A6 had good catalytic activity toward marker substrate coumarin in a reconstituted system (K(m) = 1.48 +/- 0.37 microM, V(max) = 3.36 +/- 0.18 nmol product/min/nmol CYP). Its activity in the reconstituted system was stimulated by the presence of cytochrome b(5) and glutathione. CYP2A6 was shown to metabolize chlorzoxazone in the reconstituted system with activity of 0.32 nmol of product/min/nmol of CYP, and thus caution should be taken when interpretation of CYP2E1 in vivo phenotyping data is performed. Rabbit polyclonal antibodies were produced against recombinant CYP2A6 and proved to be very useful for immunoblotting and immunoinhibition studies. Availability of this expression system and specific antibodies should facilitate characterization of the role of CYP2A6 in the metabolism of chemicals and in the study biological relevance of genetic polymorphisms of this enzyme.
Soucek
P
P
Center of Occupational Diseases, National Institute of Public Health, Srobárova 48, Praha 10, 100 42, Czech Republic. pavel.soucek@ecn.cz
D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry
D017931:DNA Primers; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6