Abstract
A procedure is outlined that is used for purification of heterologously expressed P4503A4 from Escherichia coli membranes. Details of constructron of the particular plasmid expressed (NF14) are presented elsewhere (1, 2). The general procedure can be utilized for other cytochromes P450, with some modification, as described elsewhere (1, 3–7). Several general purification techniques are described in detarl in Chapters 4 and 5 of this volume.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Gillam, E. M. J., Baba, T., Kim, B-R., Ohmori, S., and Guengerich, F. P (1993) Expression of modified human cytochrome P4503A4 in Escherichia coli and purification and reconstitution of the enzyme. Arch Biochem Biophys. 305, 123–131
Guengerich, F P., Martin, M. V., Guo, Z., and Chun, Y-J (1996) Purification of recombinant human cytochrome P450 enzymes expressed in bacteria Methods Enzymol. 272, 35–44.
Sandhu, P., Baba, T., and Guengerich, F. P (1993) Expression of modified cytochrome P4502C10 (2C9) in Escherichia coli, purification, and reconstitution of catalytic activity. Arch Biochem. Biophys. 306, 443–450.
Sandhu, P., Guo, Z., Baba, T., Martin, M. V, Tukey, R. H., and Guengerich, F. P (1994) Expression of modified human cytochrome P4501A2 in Escherichia coli: stabilization, purification, spectral characterization, and catalytic activities of the enzyme Arch Biochem Biophys 309, 168–177
Gillam, E. in J., Guo, Z, and Guengench, F. P. (1994) Expression of modified human cytochrome P4502El in Escherichia coli, purification, and spectral and catalytic properties Arch Biochem Biophys 312, 59–66
Gillam, E. M. J., Guo, Z., Ueng, Y-F., Yamazaki, H., Cock, I., Reilly, P E B, Hooper, W. D., and Guengerich, F P. (1995) Expression of cytochrome P4503A5 in Escherichia coli. effects of 5’ modifications, purification, spectral characterization, reconstitution conditions, and catalytic activities Arch Biochem Biophys 317, 374–384.
Guo, Z., Gillam, E. M. J., Ohmori, S., Tukey, R. H., and Guengerich, F. P (1994) Expression of modified human cytochrome P4501A1 in Escherichia coli effects of 5’ substitution, stabilization, purification, spectral characterization, and catalytic properties. Arch Biochem Biophys 312, 436–446.
Omura, T. and Sato, R. (1964) The carbon monoxide-binding pigment of liver mmrosomes, I. Evidence for its hemoprotein nature. J Biol Chem. 239, 2370–2378
Guengerich, F P (1994) Analysis and characterization of enzymes, in Principles and Methods of Toxicology (3rd ed.) (Hayes, A. W., ed.), Raven, NY, pp. 1259–1313.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
Bordier, C. (1981) Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem 256, 1604–1607
Sanchez-Ferrer, A., Bru, R., and Garcia-Carmona, F (1994) Phase separation of biomolecules in polyoxyethylene glycol noniomc detergents Crit Rev Blochem Mel Biol 29, 275–313.
Jenkms, C M. and Waterman, M R. (1994) Flavodoxin and NADPH-flavodoxm reductase from Escherichia coli support bovine cytochrome P45Oc17 hydroxylase activities J Biol Chem. 269, 27,401–27,408
Gillam, E. M. J., Guo, Z., Martin, M. V, Jenkins, C. M., and Guengerich, F P (1995) Expression of cytochrome P4502D6 in Escherichia coli, purification, and spectral and catalytic characterization. Arch Blochem Biophys 319, 540–550
Halkier, B A., Nielsen, H. L, Koch, B, and Moller, B L. (1995) Purification and characterizatton of recombinant cytochrome P450TYR expressed at high levels in Escherichia co1i Arch. Biochem Biophys 322, 369–377
Porath, J. (1992) Immobilized metal ton affinity chromatography. Protean Express Purif 3, 263–281
Porath, J., Carlsson, J, Olsson, I., and Belfrage, G. (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258, 598–599
Imai, T., Globerman, H., Gertner, J. in, Kagawa, N., and Waterman, M R. (1993) Expression and purification of functional human 17α-hydroxylasell7,20-lyase (P450c17) in Escherichia coli Use of this system for study of a novel form of combined 17α-hydroxylasel/7,20-lyase deficiency. J Biol Chem 268, 19,681–19,689
Kempf, A., Zanger, U M., and Meyer, U. A (1995) Truncated human P4502D6: expression in Escherichia coli Ni2+-chelate affinity purification, and characterization of solubility and aggregation Arch Biochem Biophys. 321, 277–288
Fulco, A. J and Ruettinger, R. T. (1987) Occurrence of a barbiturate-mducible catalytically self-sufficient 119,000 Dalton cytochrome P-450 monooxygenase in Bacilli. Life SCI. 40, 1769–1775.
White, K. A., and Marletta, M. A. (1992) Nitric oxide synthase is a cytochrome P-450 type hemoprotein. Biochemistry 31, 6627–6631.
McMillan, K, Bredt, D S., Hirsch, D. J, Snyder, S H., Clark, J E., and Masters, B S. S. (1992) Cloned, expressed rat cerebellar nitric oxide synthase contains stoichiometric amounts of heme, which binds carbon monoxide Proc Nut1 Acad Sci USA 89, 11,141–11,145
Murakami, H, Yabusaki, Y., Sakaki, T., Shibata, M., and Ohkawa, H. (1987) A genetically engineered P450 monooxygenase. construction of the functional fused enzyme between rat cytochrome P450c and NADPH-cytochrome P450 reductase DNA 6, 189–197.
Fisher, C W., Shet, M. S., Caudle, D. L., Martin-Wixtrom, C. A, and Estabrook, R. W (1992) High-level expression in Escherichia coli of enzy-matically active fusion proteins containing the domains of mammalian cytochromes P450 and NADPH-P450 reductase flavoprotem Proc Nat1 Acad Set USA 89, 10,817–10,821
Shet, M. S., Fisher, C. W., Arlotto, M. P., Shackleton, C. H L, Holmans, P. L., Martin-Wixtrom, C. A, Saeki, Y., and Estabrook, R. W (1994) Purification and enzymatic properties of a recombmant fusion protem expressed in Escherichia coli contaming the domains of bovine P45017A and rat NADPH-P450 reductase. Arch Biochem Biophys 311, 402–417.
Chun, Y-J., Slumada, T, and Guengerich, F. P (1996) Construction of a human cytochrome P450 l Al:rat NADPH-P450 reductase fusion protein cDNA, expres-sion in Escherichia coli, purification, and catalytic propertles of the enzyme in bacterial cells and after purification Arch Blochem Biophys 330, 48–58.
Pankh, A. and Guengench, F. P. (1996) Expression, purification, and character-ization of a catalytically active human cytochrome P450 lA2’NADPH-cyto-chrome P450 reductase fusion protein. Protean Express. Purif 9, 346–354.
Alterman, M. A., Chaurasla, C. S., Lu, P, Hardwick, J. P, and Hanzhk, R P (1995) Fatty acid dlscrlmmatlon and -hydroxylatlon by cytochrome P45404Al and a cytochrome P4504Al/NADPH-P450 reductase fusion protein. Arch Blochem Biophys 320, 289–296
Vaz, A D N, Pernecky, S J, Raner, G in, and Coon, M J (1996) Peroxo-iron and oxenmd-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: swltchmg by threonme-302 to alanine mutagenesis of cyto-chrome P4502B4 Proc Nat1 Acad. Sci. USA 93, 4644–4648.
Smith, D. B. and Johnson, K. S. (1988) Single-step purification of protems expressed in Escherichia coli as fusions with glutathione S-transferase Gene 67, 31–40
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1998 Humana Press Inc.
About this protocol
Cite this protocol
Peter Guengerich, F., A. Hosea, N., V. Martin, M. (1998). Purification of Cytochromes P450: Products of Bacterial Recombinant Expression Systems. In: Phillips, I.R., Shephard, E.A. (eds) Cytochrome P450 Protocols. Methods in Molecular Biology™, vol 107. Humana Press. https://doi.org/10.1385/0-89603-519-0:77
Download citation
DOI: https://doi.org/10.1385/0-89603-519-0:77
Publisher Name: Humana Press
Print ISBN: 978-0-89603-519-5
Online ISBN: 978-1-59259-580-8
eBook Packages: Springer Protocols