RESUMO
The expression levels of cytochrome P450 2B4 variants with N- and C-terminal modifications were compared and some of the enzymatic characteristics of recombinant proteins studied. Following C-terminal hybrids for CYP2B4 gene were constructed: 1) with intein-chitin binding domain cassette 2) with hexahistidine tag. These modifications were combined with P450 2B4 glutathione-S-transferase N-terminal fusions [Pernecky S.J., et. al., (1995) Arch. Biochem. Biophys., 318, 446-456]. The obtained constructs provided for the synthesis of full-length protein products in E. coli cells with holoenzyme yield at the levels of 200-1000 nmoles/l of the bacterial culture. Partial in vivo proteolysis was observed for C-terminal fusions with intein moiety despite the presence of glycine aminoacid residue at the junction of two proteins. The principle inapplicability of standard purification scheme for isolation of P450 2B4-intein fusions is demonstrated, since the P450 domain is inactivated at 40 mM DTT concentrations. The recombinant full-length CYP 2B4 with C-terminal oligohistidine tail was expressed under the control of T7 promoter and purified using immobilized metal-ion chelating chromatography. The C-terminal hexahistidine tag does not affect the catalytic properties of recombinant enzyme in 7-pentoxyresorufin O-dealkylation reaction.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/metabolismo , Escherichia coli/metabolismo , Esteroide Hidroxilases/metabolismo , Western Blotting , Cromatografia de Afinidade , Sistema Enzimático do Citocromo P-450/genética , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase/genética , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Fluorescência , Esteroide Hidroxilases/genéticaRESUMO
Human cytochrome P450 2B6 gene from plasmid pUC9, carrying cytochrome CYP2B6 cDNA was cloned into eucariotic expression vector pcDNA 3.1 (+). Cytochrome P450 2B6 gene in recombinant plasmid pcDNA 3.1 (+)/CYP 2B6 was expressed in E. coli cells. The expression of catalytically active recombinant protein was 60-100 nm per liter of the culture medium.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/enzimologia , Oxirredutases N-Desmetilantes/genética , Clonagem Molecular , Citocromo P-450 CYP2B6 , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Humanos , Oxirredutases N-Desmetilantes/química , Oxirredutases N-Desmetilantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
This paper describes the modification of the method by Coon and Pernecky (Meth. Enzymol. 1996, 272, 25-34) for purification of truncated (delta 2-27) recombinant form of cytochrome P450 2B4 expressed in E. coli as fusion protein with glutathione-S-transferase. The modifications included optimisation of conditions for proteolytic reaction of fusion protein with thrombine, removal of this protease from purified cytochrome P450 preparations using column chromatography on hydroxyapatite, introduction of the additional step for obtaining of spheroplasts using of lysozyme, and optimisation of conditions for enzyme stabilisation during of its purification and storage. The overall yield of purified cytochrome was 20% and the specific content of P450 was 14,5 nmol/mg protein was measured. This method is suitable for large-scale isolation of high purified cytochromes P450 which are necessary for study of structure-functional relationships of this hemoprotein with protein partners as well as for investigation of its structure and mechanism of action.