Voprosy meditsinskoi khimii (ISSN 0042-8809)

Isolation and purification of truncated (*2-27) recombinant formof cytochrome P450 2B4 expressed in E.coli as fusion protein with glutathione-s-transferase

   
Sokolov N.N., Alexandrova S.A., Omelyanuk N.M., Kirsanova I.D., Chupirina I.V., Solodar L.I., Archakov A.I.
PubMed Id: 10205825
Year: 1999 vol: 45  issue:1  pages: 24-29
Abstract: This paper describes the modification of the method by Coon andPernecky (Meth. Enzymol. 1996, 272, 25-34) for purification oftruncated (*2-27) recombinant form of cytochrome P450 2B4expressed in E.coli as fusion protein withglutathione-S-transferase. The modifications includedoptimisation of conditions for proteolytic reaction of fusionprotein with thrombine, removal of this protease from purifiedcytochrome P450 preparations using column chromatography onhydroxyapatite, introduction of the additional step for obtainingof spheroplasts using of lysozyme, and optimisation of conditionsfor enzyme stabilisation during of its purification and storage.The overall yield of purified cytochrome was 20% and the specificcontent of P450 was 14,5 nmol/mg protein was measured. Thismethod is suitable for large-scale isolation of high purifiedcytochromes P450 which are necessary for study ofstructure-functional relationships of this hemoprotein withprotein partners as well as for investigation of its structureand mechanism of action.
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Reference: Sokolov N.N., Alexandrova S.A., Omelyanuk N.M., Kirsanova I.D., Chupirina I.V., Solodar L.I., Archakov A.I., Isolation and purification of truncated (*2-27) recombinant formof cytochrome P450 2B4 expressed in E.coli as fusion protein with glutathione-s-transferase, Voprosy meditsinskoi khimii, 1999, vol: 45(1), 24-29.
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