Preparation of a biologically active apo-cytochrome b5 via heterologous expression in Escherichia coli.
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 DH5 alpha 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 beta-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).
Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 43 Prague 2, Czech Republic.
Vera
V
Kotrbová
Dagmar
D
Aimová
Marek
M
Ingr
Lucie
L
Borek-Dohalská
Václav
V
Martínek
Marie
M
Stiborová
D001051:Apoenzymes / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009284:Naphthols / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D001051:Apoenzymes; D009284:Naphthols; D011994:Recombinant Proteins; D006418:Heme; C024336:1-phenylazo-2-naphthol; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human