Heterologous expression of eukaryotic cytochromes P450 1. Heterologous expressionof cytochrome P450 2B4 in e.coli as fusion proteins with different affiniti purificationtags
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.
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Jgoun A.A., Eldarov M.A., Solodar L.I., Sokolov N.N., Archakov A.I., Skryabin K.G. (2001) Heterologous expression of eukaryotic cytochromes P450 1. Heterologous expressionof cytochrome P450 2B4 in e.coli as fusion proteins with different affiniti purificationtags. Voprosy Meditsinskoi Khimii, 47(4), 382-392.
Jgoun A.A. et al. Heterologous expression of eukaryotic cytochromes P450 1. Heterologous expressionof cytochrome P450 2B4 in e.coli as fusion proteins with different affiniti purificationtags // Voprosy Meditsinskoi Khimii. - 2001. - V. 47. -N 4. - P. 382-392.
Jgoun A.A. et al., "Heterologous expression of eukaryotic cytochromes P450 1. Heterologous expressionof cytochrome P450 2B4 in e.coli as fusion proteins with different affiniti purificationtags." Voprosy Meditsinskoi Khimii 47.4 (2001): 382-392.
Jgoun, A. A., Eldarov, M. A., Solodar, L. I., Sokolov, N. N., Archakov, A. I., Skryabin, K. G. (2001). Heterologous expression of eukaryotic cytochromes P450 1. Heterologous expressionof cytochrome P450 2B4 in e.coli as fusion proteins with different affiniti purificationtags. Voprosy Meditsinskoi Khimii, 47(4), 382-392.
References
Porter T.D. and Larson J.R. (1991) Meth.Enzymol. 206,108- 111. Scholar google search
Jgoun A.A., Sidorovich V.E., Eldarov M.A. et al. (1998) 12th InternationalSymposium on MDO, Montpeller, abstract 131. Scholar google search
Prozorovsky Т., Schuck W.H., Archakov A.I. (2000) International workshop "From sequence to function: experimental and bioinformatic studies of cytpchome P450 superfamily", Moscow, p.62. Scholar google search
