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. 1991 Jul 1;88(13):5597–5601. doi: 10.1073/pnas.88.13.5597

Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

H J Barnes 1, M P Arlotto 1, M R Waterman 1
PMCID: PMC51924  PMID: 1829523

Abstract

When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Asseffa A., Smith S. J., Nagata K., Gillette J., Gelboin H. V., Gonzalez F. J. Novel exogenous heme-dependent expression of mammalian cytochrome P450 using baculovirus. Arch Biochem Biophys. 1989 Nov 1;274(2):481–490. doi: 10.1016/0003-9861(89)90461-x. [DOI] [PubMed] [Google Scholar]
  2. Battula N., Sagara J., Gelboin H. V. Expression of P1-450 and P3-450 DNA coding sequences as enzymatically active cytochromes P-450 in mammalian cells. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4073–4077. doi: 10.1073/pnas.84.12.4073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Estabrook R. W., Mason J. I., Martin-Wixtrom C., Zuber M., Waterman M. R. Some enzymatic vagaries of a bovine adrenal microsomal cytochrome P-450 introduced and expressed in transformed monkey kidney cells. Prog Clin Biol Res. 1988;274:525–540. [PubMed] [Google Scholar]
  4. Fujiki Y., Hubbard A. L., Fowler S., Lazarow P. B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol. 1982 Apr;93(1):97–102. doi: 10.1083/jcb.93.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gegner J. A., Dahlquist F. W. Signal transduction in bacteria: CheW forms a reversible complex with the protein kinase CheA. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):750–754. doi: 10.1073/pnas.88.3.750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Higuchi R., Krummel B., Saiki R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. doi: 10.1093/nar/16.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Larson J. R., Coon M. J., Porter T. D. Alcohol-inducible cytochrome P-450IIE1 lacking the hydrophobic NH2-terminal segment retains catalytic activity and is membrane-bound when expressed in Escherichia coli. J Biol Chem. 1991 Apr 25;266(12):7321–7324. [PubMed] [Google Scholar]
  8. Looman A. C., Bodlaender J., Comstock L. J., Eaton D., Jhurani P., de Boer H. A., van Knippenberg P. H. Influence of the codon following the AUG initiation codon on the expression of a modified lacZ gene in Escherichia coli. EMBO J. 1987 Aug;6(8):2489–2492. doi: 10.1002/j.1460-2075.1987.tb02530.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Loose D. S., Kan P. B., Hirst M. A., Marcus R. A., Feldman D. Ketoconazole blocks adrenal steroidogenesis by inhibiting cytochrome P450-dependent enzymes. J Clin Invest. 1983 May;71(5):1495–1499. doi: 10.1172/JCI110903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Masters B. S., Okita R. T. The history, properties, and function of NADPH-cytochrome P-450 reductase. Pharmacol Ther. 1980;9(2):227–244. doi: 10.1016/s0163-7258(80)80020-9. [DOI] [PubMed] [Google Scholar]
  11. McClelland M., Hanish J., Nelson M., Patel Y. KGB: a single buffer for all restriction endonucleases. Nucleic Acids Res. 1988 Jan 11;16(1):364–364. doi: 10.1093/nar/16.1.364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Muchmore D. C., McIntosh L. P., Russell C. B., Anderson D. E., Dahlquist F. W. Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance. Methods Enzymol. 1989;177:44–73. doi: 10.1016/0076-6879(89)77005-1. [DOI] [PubMed] [Google Scholar]
  13. Nakajin S., Shinoda M., Haniu M., Shively J. E., Hall P. F. C21 steroid side chain cleavage enzyme from porcine adrenal microsomes. Purification and characterization of the 17 alpha-hydroxylase/C17,20-lyase cytochrome P-450. J Biol Chem. 1984 Mar 25;259(6):3971–3976. [PubMed] [Google Scholar]
  14. Narasimhulu S., Cooper D. Y., Rosenthal O. Spectrophotometric properties of a Triton-clarified steroid 21-hydroxylase system of adrenocortical microsomes. Life Sci. 1965 Nov;4(21):2101–2107. doi: 10.1016/0024-3205(65)90328-0. [DOI] [PubMed] [Google Scholar]
  15. Narhi L. O., Fulco A. J. Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium. J Biol Chem. 1986 Jun 5;261(16):7160–7169. [PubMed] [Google Scholar]
  16. Nebert D. W., Nelson D. R., Coon M. J., Estabrook R. W., Feyereisen R., Fujii-Kuriyama Y., Gonzalez F. J., Guengerich F. P., Gunsalus I. C., Johnson E. F. The P450 superfamily: update on new sequences, gene mapping, and recommended nomenclature. DNA Cell Biol. 1991 Jan-Feb;10(1):1–14. doi: 10.1089/dna.1991.10.1. [DOI] [PubMed] [Google Scholar]
  17. OMURA T., SATO R. A new cytochrome in liver microsomes. J Biol Chem. 1962 Apr;237:1375–1376. [PubMed] [Google Scholar]
  18. Oeda K., Sakaki T., Ohkawa H. Expression of rat liver cytochrome P-450MC cDNA in Saccharomyces cerevisiae. DNA. 1985 Jun;4(3):203–210. doi: 10.1089/dna.1985.4.203. [DOI] [PubMed] [Google Scholar]
  19. Osborn M. J., Munson R. Separation of the inner (cytoplasmic) and outer membranes of Gram-negative bacteria. Methods Enzymol. 1974;31:642–653. doi: 10.1016/0076-6879(74)31070-1. [DOI] [PubMed] [Google Scholar]
  20. Ostrowski J., Barber M. J., Rueger D. C., Miller B. E., Siegel L. M., Kredich N. M. Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequence deduced from DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase. J Biol Chem. 1989 Sep 25;264(27):15796–15808. [PubMed] [Google Scholar]
  21. Porter T. D., Wilson T. E., Kasper C. B. Expression of a functional 78,000 dalton mammalian flavoprotein, NADPH-cytochrome P-450 oxidoreductase, in Escherichia coli. Arch Biochem Biophys. 1987 Apr;254(1):353–367. doi: 10.1016/0003-9861(87)90111-1. [DOI] [PubMed] [Google Scholar]
  22. Ruettinger R. T., Wen L. P., Fulco A. J. Coding nucleotide, 5' regulatory, and deduced amino acid sequences of P-450BM-3, a single peptide cytochrome P-450:NADPH-P-450 reductase from Bacillus megaterium. J Biol Chem. 1989 Jul 5;264(19):10987–10995. [PubMed] [Google Scholar]
  23. Sakaki T., Shibata M., Yabusaki Y., Murakami H., Ohkawa H. Expression of bovine cytochrome P450c17 cDNA in Saccharomyces cerevisiae. DNA. 1989 Jul-Aug;8(6):409–418. doi: 10.1089/dna.1.1989.8.409. [DOI] [PubMed] [Google Scholar]
  24. Schauder B., McCarthy J. E. The role of bases upstream of the Shine-Dalgarno region and in the coding sequence in the control of gene expression in Escherichia coli: translation and stability of mRNAs in vivo. Gene. 1989 May 15;78(1):59–72. doi: 10.1016/0378-1119(89)90314-4. [DOI] [PubMed] [Google Scholar]
  25. Stormo G. D., Schneider T. D., Gold L. M. Characterization of translational initiation sites in E. coli. Nucleic Acids Res. 1982 May 11;10(9):2971–2996. doi: 10.1093/nar/10.9.2971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Unger B. P., Gunsalus I. C., Sligar S. G. Nucleotide sequence of the Pseudomonas putida cytochrome P-450cam gene and its expression in Escherichia coli. J Biol Chem. 1986 Jan 25;261(3):1158–1163. [PubMed] [Google Scholar]
  27. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  28. Zuber M. X., John M. E., Okamura T., Simpson E. R., Waterman M. R. Bovine adrenocortical cytochrome P-450(17 alpha). Regulation of gene expression by ACTH and elucidation of primary sequence. J Biol Chem. 1986 Feb 15;261(5):2475–2482. [PubMed] [Google Scholar]
  29. Zuber M. X., Simpson E. R., Hall P. F., Waterman M. R. Effects of adrenocorticotropin on 17 alpha-hydroxylase activity and cytochrome P-450(17 alpha) synthesis in bovine adrenocortical cells. J Biol Chem. 1985 Feb 10;260(3):1842–1848. [PubMed] [Google Scholar]
  30. Zuber M. X., Simpson E. R., Waterman M. R. Expression of bovine 17 alpha-hydroxylase cytochrome P-450 cDNA in nonsteroidogenic (COS 1) cells. Science. 1986 Dec 5;234(4781):1258–1261. doi: 10.1126/science.3535074. [DOI] [PubMed] [Google Scholar]

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