[Heterologous expression of eukaryotic CYP450. 1. Heterologous expression of cytochrome P450 2B4 using groups with various affinity in E. coli].
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.
Zhgun
A A
AA
Centre Bioengineering RAS, Prospect 60-letya Octyabrya, 7/1, Moscow 117571.
El'darov
M A
MA
Solodar'
L I
LI
Sokolov
N N
NN
Archakov
A I
AI
Skriabin
K G
KG
D001189:Aryl Hydrocarbon Hydroxylases; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C050356:steroid 15-alpha-hydroxylase; D005982:Glutathione Transferase