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 ac hieve expression, the N-terminus of protein was modified by PCR mutagenesis . The N-terminal variant with only a single amino acid change showed expres sion of 210 nmol of CYP2A6/liter of culture, Recombinant CYP2A6 protein was purified to electrophoretic homogeneity and further characterized. Absolut e spectra were typical for CYP proteins and indicated low spin characterist ics of isolated protein. Due to a hydrophobic segment the N-terminal amino acid sequence of recombinant CYP2A6 was blocked. The N-terminal formyl-meth ionine block was removed by mild acid treatment. Purified CYP2A6 had good c atalytic activity toward marker substrate coumarin in a reconstituted syste m (K-m = 1.48 +/- 0.37 mu M, V-max = 3.36 +/- 0.18 nmol product/min/nmol CY P). Its activity in the reconstituted system was stimulated by the presence of cytochrome b(5) and glutathione. CYP2A6 was shown to metabolize chlorzo xazone in the reconstituted system with activity of 0.32 nmol of product/mi n/nmol of CYP, and thus caution should be taken when interpretation of CYP2 E1 in vivo phenotyping data is performed. Rabbit polyclonal antibodies were produced against recombinant CYP2A6 and proved to be very useful for immun oblotting and immunoinhibition studies. Availability of this expression sys tem and specific antibodies should facilitate characterization of the role of CYP2A6 the metabolism of chemicals and in the study biological relevance of genetic polymorphisms of C his enzyme. (C)1999 Academic Press.