Voprosy meditsinskoi khimii (ISSN 0042-8809)

Heterologous expression of eukaryotic cytochromes P450 1. Heterologous expressionof cytochrome P450 2B4 in e.coli as fusion proteins with different affiniti purificationtags

   


1. Centre "Bioengineering"RAS
2. Orekhovich Institute of Biomedical Chemistry RAMS
PubMed Id: 11693026
Year: 2001 vol: 47  issue:4  pages: 382-392
Abstract: The expression levels of cytochrome P450 2B4 variants with N- and C-terminalmodifications were compared and some of the enzymatic characteristics of recombinantproteins studied. Following C-terminal hybrids for CYP2B4 gene were constructed: 1) withintein-chitin binding domain cassette 2) with hexahistidine tag. These modifications werecombined 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 thesynthesis of full-length protein products in E.coli cells with holoenzyme yield at thelevels of 200-1000 nmoles/l of the bacterial culture. Partial in vivo proteolysis wasobserved for C-terminal fusions with intein moiety despite the presence of glycineaminoacid residue at the junction of two proteins. The principle inapplicability ofstandard purification scheme for isolation of P450 2B4-intein fusions is demonstrated,since the P450 domain is inactivated at 40 mM DTT concentrations. The recombinantfull-length CYP 2B4 with C-terminal oligohistidine tail was expressed under the control ofT7 promoter and purified using immobilized metal-ion chelating chromatography. TheC-terminal hexahistidine tag does not affect the catalytic properties of recombinantenzyme in 7-pentoxyresorufin O-dealkylation reaction.
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Reference: Jgoun A.A., Eldarov M.A., Solodar L.I., Sokolov N.N., Archakov A.I., Skryabin K.G., 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, vol: 47(4), 382-392.
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