[Isolation and purification of recombinant truncated (delta2-27) cytochrome P450 2B4 expressed in E. coli cells as a fusion protein with glutathione-S-transferase].
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.
Sokolov
N N
NN
Institute of Biomedical Chemistry RAMS, Moscow. sokolov@medic.ibmh.msk.su
Aleksandrova
S S
SS
Omel'ianiuk
N M
NM
Kirsanova
I D
ID
Chupyrina
I V
IV
Solodar'
L I
LI
Archakov
A I
AI
D001189:Aryl Hydrocarbon Hydroxylases; D002846:Chromatography, Affinity; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005982:Glutathione Transferase / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D012995:Solubility; D013250:Steroid Hydroxylases / Q000302:isolation & purification*
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