Purification of Cytochrome P‐450 Enzymes

L.C. Bell‐Parikh1, N.A. Hosea1, M.V. Martin1, F.P. Guengerich1

1 Vanderbilt University School of Medicine, Nashville, Tennessee
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.2
DOI:  10.1002/0471140856.tx0402s12
Online Posting Date:  August, 2002
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Abstract

Among the liver P‐450 xenobiotic‐metabolizing enzymes, P450‐2E1 is of interest because of its activation of potent carcinogens, and P‐450 1A2 is of interest because of its role in oxidation of drugs and carcinogens. This unit describes column chromatography protocols for purification of recombinant forms of these enzymes expressed in a bacterial expression system.

     
 
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Table of Contents

  • Basic Protocol 1: Column Chromatography for Purification of Recombinant Human P‐450 2E1
  • Basic Protocol 2: Column Chromatography for Purification of Recombinant Human P‐450 1A2
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Column Chromatography for Purification of Recombinant Human P‐450 2E1

  Materials
  • E. coli membrane preparation containing P‐450 2E1 (Gillam et al., )
  • Membrane solubilization solution for P‐450 2E1 (see recipe)
  • Diethylaminoethyl (DEAE) Sephacel ion exchanger resin (Pharmacia Biotech)
  • DEAE equilibration buffer for P‐450 2E1 (see recipe)
  • Carboxymethyl (CM) Sepharose Fast Flow ion exchanger resin (Pharmacia Biotech)
  • CM equilibration buffer for P‐450 2E1 (see recipe)
  • 20% (v/v) glycerol (see recipe)
  • 0.1 to 1 M H 3PO 4
  • 50 mM CM wash buffer for P‐450 2E1 (see recipe)
  • 10 mM CM elution buffer (see recipe)
  • 200 mM CM elution buffer (see recipe)
  • Hydroxylapatite (HA) Fast Flow chromatographic support (Calbiochem)
  • HA equilibration buffer (see recipe)
  • HA elution buffer (see recipe)
  • Post‐HA dialysis buffer for P‐450 2E1 (see recipe)
  • Empty 20‐ and 100‐ml chromatographic columns
  • Empty CM Sepharose Fast Flow column
  • Spectrapor 12,000‐ to 14,000‐MWCO dialysis tubing (Spectrum Medical Industries)
  • Additional reagents and equipment for ion exchange chromatography ( appendix 3A) and for SDS‐PAGE and silver staining of gels ( appendix 3A)

Basic Protocol 2: Column Chromatography for Purification of Recombinant Human P‐450 1A2

  Materials
  • E. coli membrane preparation containing P‐450 1A2 (Sandhu et al., )
  • Membrane solubilization solution for P‐450 1A2 (see recipe)
  • Diethylaminoethyl (DEAE) Sephacel Fast Flow ion exchanger resin (Pharmacia Biotech)
  • DEAE equilibration buffer for P‐450 1A2 (see recipe)
  • Carboxymethyl (CM) Sepharose Fast Flow ion exchanger resin (Pharmacia Biotech)
  • CM equilibration buffer for P‐450 1A2 (see recipe)
  • 2× dilution solution (see recipe)
  • 50 mM CM wash buffer for P‐450 1A2 (see recipe)
  • 100 mM CM wash buffer (see recipe)
  • 300 mM CM elution buffer (see recipe)
  • 500 mM CM elution buffer (see recipe)
  • Empty 40‐ and 70‐ml chromatographic columns
  • Additional reagents and solutions for ion exchange chromatography ( appendix 3A) and for SDS‐PAGE and silver staining of gels ( appendix 3A)
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Literature Cited

Literature Cited
   Butler, M.A., Iwasaki, M., Guengerich, F.P., and Kadlubar, F.F. 1989. Human cytochrome P‐450PA (P‐450IA2), the phenacetin O‐deethylase, is primarily responsible for the hepatic 3‐demethylation of caffeine and N‐oxidation of carcinogenic arylamines. Proc. Natl. Acad. Sci. U.S.A. 86:7696‐7700.
   Gillam, E.M.J., Baba, T., Kim, B‐R., Ohmori, S., and Guengerich, F.P. 1993. Expression of modified human cytochrome P‐450 3A4 in Escherichia coli and purification and reconstitution of the enzyme. Arch. Biochem. Biophys. 305:123‐131.
   Gillam, E.M.J., Guo, Z., and Guengerich, F.P. 1994. Expression of modified human cytochrome P‐450 2E1 in Escherichia coli, purification, and spectral and catalytic properties. Arch. Biochem.Biophys. 312:59‐66.
   Gillam, E.M.J., Guo, Z., Martin, M.V., Jenkins, C.M., and Guengerich, F.P. 1995. Expression of cytochrome P‐450 2D6 in Escherichia coli, purification, and spectral and catalytic characterization. Arch. Biochem. Biophys. 319:540‐550.
   Grogan, J., Shou, M., Andrusiak, E.A., Tamura, S., Buters, J.T.M., Gonzalez, F.J., and Korzekwa, K.R. 1995. Cytochrome P‐450 2A1, 2E1, and 2C9 cDNA‐expression by insect cells and partial purification using hydrophobic chromatography. Biochem. Pharmacol. 50:1509‐1515.
   Guengerich, F.P. 1987. Mammalian cytochromes P‐450 Vol. 1. CRC Press, Boca Raton, Fla.
   Guengerich, F.P. 1994. Analysis and characterization of enzymes. In Principles and Methods of Toxicology, 3rd ed. (Hayes, A.W., ed.) pp. 1259‐1313. Raven Press, New York.
   Guengerich, F.P. 1995. Human cytochrome P‐450 enzymes. In Cytochrome P‐450, 2nd ed. (Ortiz de Montellano, P.R., ed.) pp. 473‐535. Plenum Press, New York.
   Guengerich, F.P., Martin, M.V., Guo, Z., and Chun, Y.J. 1996. Purification of functional recombinant P‐450s from bacteria. Methods. Enzymol. 272:35‐44.
   Imaoka, S., Yamada, T., Hiroi, T., Hayashi, K., Sakaki, T., Yabusaki, Y., and Funae, Y. 1996. Multiple forms of human P‐450 expressed in Saccharomyces cerevisiae: Systematic characterization and comparison with those of the rat. Biochem. Pharmacol. 51:1041‐1050.
   Josephy, P.D., DeBruin, L.S., Lord, H.L., Oak, J., Evans, D.H., Guo, Z., Dong, M.‐S., and Guengerich, F.P. 1995. Bioactivation of aromatic amines by recombinant human cytochrome P‐450 1A2 expressed in bacteria: A substitute for mammalian tissue preparations in mutagenicity testing. Cancer Res. 55:799‐802.
   Omata, Y., Robinson, R.C., Gelboin, H.V., Pincus, M.R., and Friedman, F.K. 1994. Specificity of the cytochrome P‐450 interaction with cytochrome b5. FEBS Lett. 346:241‐245.
   Omura, T. and Sato, R. 1964. The carbon monoxide‐binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239:2370‐2378.
   Parikh, A. and Guengerich, F.P. 1997. Expression, purification, and characterization of a catalytically active human cytochrome P‐450 1A2:NADPH‐cytochrome P‐450 reductase fusion protein. Protein Express. Purif. 9:346‐354.
   Peter, R., Böcker, R.G., Beaune, P.H., Iwasaki, M., Guengerich, F.P. and Yang, C.S. 1990. Hydroxylation of chlorzoxazone as a specific probe for human liver cytochrome P‐450 IIE1. Chem. Res. Toxicol. 3:566‐573.
   Porter, T.D., Pernecky, S.J., Larson, J.R., Fujita, V.S., and Coon, M.J. 1990. Expression of cytochrome P‐450 in yeast and Escherichia coli. In Drug Metabolizing Enzymes: Genetics, Regulation and Toxicology, Proceedings of the Eighth International Symposium on Microsomes and Drug Oxidations (Stockholm, June 25‐29), (M. Ingelman‐Sundberg, J.‐Å. Gustafsson, and S. Orrenius, eds.) p. 20. Karolinska Institute, Stockholm.
   Sandhu, P., Guo, Z., Baba, T., Martin, M.V., Tukey, R.H. and Guengerich, F.P. 1994. Expression of modified human cytochrome P‐450 1A2 in Escherichia coli: Stabilization, purification, spectral characterization, and catalytic activities of the enzyme. Arch Biochem. Biophys. 309:168‐177.
   Waterman, M.R., and Johnson, E.F. eds. 1991. Methods in Enzymology, Vol. 206: Cytochrome P‐450. Academic Press, San Diego.
Key References
   Gillam et al., See above.
  Describes a high‐yield expression system for P‐450 2E1, as well as a procedure for isolating the P‐450‐containing membrane fraction from whole cells.
   Sandhu et al., See above.
  Describes a high‐yield expression system for P‐450 1A2.
   Guengerich, See above.
  Cites references that describe specific purification procedures for several of the major human cytochrome P‐450s, reviews recombinant technology as a source of P‐450s, and concisely summarizes the biological significance of major human P‐450 isoforms.
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