Preparation of a biologically active apo-cytochrome b5 via heterologous expression in Escherichia coli.

Abstract Cytochrome b 5 (b 5 ) has been shown to modulate many cytochrome P450 (CYP)-dependent reactions. In order to elucidate the mechanism of such modulations, it is necessary to evaluate not only the effect of native b 5 on CYP-catalyzed reactions, but also that of the apo-cytochrome b 5 (apo-b 5 ). Therefore, the apo-b 5 protein was prepared using a heterologous expression in Escherichia coli. The gene for rabbit b 5 was constructed from synthetic oligonucleotides using polymerase chain reaction (PCR), cloned into pUC19 plasmid and amplified in DH5α cells. The gene sequence was verified by DNA sequencing. The sequence coding b 5 was cleaved from pUC19 by NdeI and XhoI restriction endonucleases and subcloned to the expression vector pET22b. This vector was used to transform E. coli BL-21 (DE3) Gold cells by heat shock. Expression of b 5 was induced with isopropyl β- d -1-thiogalactopyranoside (IPTG). The b 5 protein, produced predominantly in its apo-form, was purified from isolated membranes of E. coli cells by chromatography on a column of DEAE–Sepharose. Using such procedures, the homogenous preparation of apo-b 5 protein was obtained. Oxidized and reduced forms of the apo-b 5 reconstituted with heme exhibit the same absorbance spectra as native b 5 . The prepared recombinant apo-b 5 reconstituted with heme can be reduced by NADPH:CYP reductase. The reconstituted apo-b 5 is also fully biologically active, exhibiting the comparable stimulation effect on the CYP3A4 enzymatic activity towards oxidation of 1-phenylazo-2-hydroxynaphthalene (Sudan I) as native rabbit and human b 5 .