Preparation of a biologically active apo-cytochrome b sub(5) via heterologous expression in Escherichia coli

• Published in: Protein expression and purification Vol. 66; no. 2; pp. 203 - 209
• Main Authors: Kotrbova, Vera, Aimova, Dagmar, Ingr, Marek, Borek-Dohalska, Lucie, Martinek, Vaclav, Stiborova, Marie
• Format: Journal Article
• Language: English
• Published: 01.08.2009
• Subjects: Escherichia coli
• ISSN: 1046-5928
• DOI: 10.1016/j.pep.2009.03.011

Cytochrome b sub(5) (b sub(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 sub(5) on CYP-catalyzed reactions, but also that of the apo-cytochrome b sub(5) (apo-b sub(5)). Therefore, the apo-b sub(5) protein was prepared using a heterologous expression in Escherichia coli. The gene for rabbit b sub(5) was constructed from synthetic oligonucleotides using polymerase chain reaction (PCR), cloned into pUC19 plasmid and amplified in DH5a cells. The gene sequence was verified by DNA sequencing. The sequence coding b sub(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 sub(5) was induced with isopropyl b-d-1-thiogalactopyranoside (IPTG). The b sub(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 sub(5) protein was obtained. Oxidized and reduced forms of the apo-b sub(5) reconstituted with heme exhibit the same absorbance spectra as native b sub(5). The prepared recombinant apo-b sub(5) reconstituted with heme can be reduced by NADPH:CYP reductase. The reconstituted apo-b sub(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 sub(5).