High-Level Expression of Human Cytochrome P450 3A4 by Co-Expression with Human Molecular Chaperone HDJ-1 (Hsp40)

  • Ahn, Tae-Ho (Department of Biochemistry, College of Veterinary Medicine, Chonnam National University) ;
  • Yun, Chul-Ho (School of Biological Sciences and Technoogy, Chonnam National University)
  • Published : 2004.03.01

Abstract

Cytochrome P450 (CYP) 3A4 is of great interest because of its important roles in the oxidation of numerous drugs and xenobiotics. HDJ-1, a molecular chaperone in human, is known to assist the correct folding of unfolded proteins. To achieve a high yield of recombinant human CYP3A4 in Escherichia coli, the CYP3A4 encoding gene was co-expressed with the chaperone HDJ-1, under the control of an inducible tac promoter in a bicistronic format. The levels of expression of the CYP3A4 in the bicistronic construct reached up to 715 nmol $(liter culture)^{-1}$ within 16 h at $37^{\circ}C$, which was about a 3.3-fold increase compared to that of the CYP3A4 alone without the HDJ-1. By co-expression with HDJ-1, the catalytic activity of CYP3A4 was also increased by -15-fold. The amount of activity increase was similar to that of the CYP production at the whole cell level. The present over-expression system may be useful for the rapid production of large amounts of active CYP3A4 in E. coli.

Keywords

References

  1. Arlotto, M. P., Trant, J. M., and Estabrook, R. W., Measurement of steroid hydroxylation reactions by high-performance liquid chromatography as indicator of P450 identity and function. Methods Enzymol., 206, 454-462 (1991) https://doi.org/10.1016/0076-6879(91)06114-I
  2. Bao, Y. P., Cook, L. J., ODonovan,D., Uyama,E., and Rubinsztein, D. C., Mammalian, yeast, bacterial,and chemical chaperones reduce aggregate formation and death in a cell model of oculopharyngeal muscular dystrophy. J. Biol. Chem., 277, 12263-12269 (2002) https://doi.org/10.1074/jbc.M109633200
  3. Distlerath, L. M., Reilly, P. E., Martin, M. V., Davis, G. G., Wilkinson, G. R., and Guengerich, F. P., Purification and characterization of the human liver cytochromes P-450 involved in debrisoquine 4-hydroxylation and phenacetin O-deethylation, two prototypes for genetic polymorphism in oxidative drug metabolism. J. Biol. Chem., 260, 9057-9067 (1985)
  4. Freeman, B. C. and Morimoto, R. I., The human cytosolic molecular chaperones hsp90, hsp70 (hsc70) and hdj-1 have distinct roles in recognition of a non-native protein and protein refolding. EMBO J., 15, 2969-2979 (1996)
  5. Gillam, E. M., Baba, T., Kim, B. R., Ohmori, S., and Guengerich, F. P. Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme. Arch. Biochem. Biophys., 305, 123-131 (1993) https://doi.org/10.1006/abbi.1993.1401
  6. Guengerich, F. P. and Parikh, A., Expression of drug-metabo-lizing enzymes. Curr. Opin. Biotechnol., 8, 623-628 (1997) https://doi.org/10.1016/S0958-1669(97)80039-0
  7. Guengerich, F. P., Human cytochrome P450 enzymes. In Oritiz de Montelano, P. R. (Ed.) Cytochrome P450, 2nd ed. Plenum Press, New York, pp.473-535 (1995)
  8. Hanna, I. H., Teiber, J. F., Kokones, K. L., and Hollenberg, P. F., Role of the alanine at position 363 of cytochrome P450 2B2 in influencing the NADPH- and hydroperoxide-supported activities. Arch. Biochem. Biophys., 350, 324-332 (1998) https://doi.org/10.1006/abbi.1997.0534
  9. Inoue, E., Takahashi, Y., Imai, Y., and Kamataki, T., Development of bacterial expression system with high yield of CYP3A7, a human fetus-specific form of cytochrome P450. Biochem. Biophys. Res. Commun., 269, 623-627 (2000) https://doi.org/10.1006/bbrc.2000.2340
  10. Iwata, H., Fujita, K-I., Kushida, H., Suzuki, A., Konno, Y., Nakamura, K., Fujino, A., and Kamataki, T., High catalytic activity of human cytochrome P450 co-expressed with human NADPH-cytochrome P450 reductase in Escherichia coli. Biochem. Pharmacol., 55, 1315-1325 (1998) https://doi.org/10.1016/S0006-2952(97)00643-6
  11. Nagata, H., Hansen, W. J., Freeman, B., and Welch, W. J., Mammalian cytosolic DnaJ homologues affect the hsp70 chaperone-substrate reaction cycle, but do not interact directly with nascent or newly synthesized proteins. Biochemistry, 37, 6924-6938 (1998) https://doi.org/10.1021/bi980164g
  12. Omura, T. and Sato, R., The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem., 239, 2370-2378 (1964)
  13. Parikh, A. and Guengerich, F. P., Expression, purification, and characterization of a catalytically active human cytochrome P450 3A4: Rat NADPH-cytochrome P450 fusion protein. Protein Expr. Purif., 9, 346-354 (1997) https://doi.org/10.1006/prep.1997.0721
  14. Pritchard, M. P., Ossentian, R., Li, D. N., Henderson, C. J., Burchell, B., Wolf, R., and Friedberg, T., A general strategy for the expression of recombinant human cytochrome P450s in Escherichia coli using bacterial signal peptides: Ex-pression of CYP3A4, CYP2A6, and CYP2E1. Arch. Biochem. Biophys., 345, 342-354 (1997) https://doi.org/10.1006/abbi.1997.0265
  15. Sandhu, P., Baba, T., and Guengerich, F. P., Expression of modified cytochrome P450 2C10 (2C9) in Escherichia coli, purification, and reconstitution of catalytic activity. Arch. Bioehem. Biophys., 306, 443-450 (1993) https://doi.org/10.1006/abbi.1993.1536
  16. Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J., and Roe, B. A., Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J. Mol. Biol., 143, 161-178 (1980) https://doi.org/10.1016/0022-2836(80)90196-5
  17. Tang, W. and Steams, R. A., Heterotropic cooperativity of cytochrome P450 3A4 and potential drug-drug interactions. Curr. Drug Metab., 2, 185-198 (2001) https://doi.org/10.2174/1389200013338658
  18. Wrighton, S. A. and Stevens, J. C., The human hepatic cytochromes P450 involved in drug metabolism. Crit. Rev. Toxcol., 22, 1-21 (1992) https://doi.org/10.3109/10408449209145319