Isolation and purification of truncated (*2-27) recombinant formof cytochrome P450 2B4 expressed in E.coli as 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.
Download PDF:
Keywords: cytochrome P450 2B4 (delta2-27), fusion proteins, heterologous expression of cytochrome P450 in E.coli, glutathion-S-transferase, thrombine
Citation:
Sokolov N.N., Alexandrova S.A., Omelyanuk N.M., Kirsanova I.D., Chupirina I.V., Solodar L.I., Archakov A.I. (1999) 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, 45(1), 24-29.
Sokolov N.N. et al. 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. - V. 45. -N 1. - P. 24-29.
Sokolov N.N. et al., "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 45.1 (1999): 24-29.
Sokolov, N. N., Alexandrova, S. A., Omelyanuk, N. M., Kirsanova, I. D., Chupirina, I. V., Solodar, L. I., Archakov, A. I. (1999). 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, 45(1), 24-29.
References
Archakov A., Bachmanova G. (1990) Cytochrome P450 and Active Oxygen London:Taylor & Francis. Scholar google search
Pernecky S.J., Larson J.R., Philpot R.M., Coon M.J. (1993). Proc. Natl. Acad. Sci. USA. 90, 2651-2655. Scholar google search
Larson J.R., Coon M.J., Porter T.D. (1991). J. Biol. Chem. 266, 7321-7324. Scholar google search
Pernecky S.J., Coon M.J. (1996). Methods in Enzymology. 272, 25-34. Scholar google search
Lehnerer M., Schulze J., Petzold A., Bernhardt R., Hlavica P. (1995). Biochem. Biophys. Acta. 1245, 107-115. Scholar google search
Scheller U., Juretzek Т., Schunck W.-H. (1996). Methods in Enzymology. 272, 65-75. Scholar google search
Guengerich F.P., Martin M.V., Guo Z., Chun Y.-J. (1996). Methods in Enzymology. 272, 35-44. Scholar google search
A.A.Zhgoon, V.E.Sidorovoch, M.A.Eldarov, E.A.Apktalina, N.N.Sokolov, W.H.Schunck, A.I.Archakov and ICG.Skryabin,. (1998). 12* International Symposium on Microsomes and Drug Oxidation, Montpellier, France, abstract 131. Scholar google search
Sambrook J.F., Fritsch E.F., Maniatis T. (1989). Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York?. Scholar google search
