Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Expression and Purification of Human Farnesoid X Receptor-Ligand Binding Domain as Soluble Form Using a Dual Cistronic Expression Vector

  • Kang, Hyun (Department of Biotechnology, College of Biomedical and Health Science, Konkuk University) ;
  • Ye, Micheal B. (Department of Applied Biochemistry, College of Biomedical and Health Science, Konkuk University) ;
  • Bahk, Young Yil (Department of Biotechnology, College of Biomedical and Health Science, Konkuk University)
  • Received : 2012.12.05
  • Accepted : 2012.12.21
  • Published : 2013.03.28
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Abstract

In this study, we show the expression and purification of the human recombinant farnesoid X receptor (FXR)- ligand binding domain (LBD) protein in E. coli using a double cistronic vector, pACYCDuet-1, as a soluble form. We describe here the expression and characterization of a biologically active $FXR-LBD_{(248-476)}$. When expressed in the influence of bacterial promoters ($P_{T7}$ and $P_{Tac}$) of the single cistronic expression vectors, the human recombinant $FXR-LBD_{(248-476)}$ was found to be totally insoluble. However, by using a double cistronic expression vector, we were able to obtain the human recombinant $FXR-LBD_{(248-476)}$ in a soluble form. To allow for biological activities, we have subcloned into the pACYCDuet-1 vector, expressed in E. coli cells at some optimized conditions, and purified and characterized the human recombinant active $FXR-LBD_{(248-476)}$ proteins using the fluorescence polarization assay. This suggests that the expression of FXR-LBD in a double cistronic vector improves its solubility and probably assists its correct folding for the biologically active form of the proteins. We suggest that this may represent a new approach to high expression of other nuclear receptors and may be useful as well for other classes of heterodimeric protein partners.

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References

  1. Altucci, L., M. D. Leibowitz, K. M. Ogilvie, A. R. de Leva, and H. Gronemeyer. 2007. RXR and RXR modulation in cancer and metabolic disease. Nat. Rev. Drug Discov. 6: 793-810. https://doi.org/10.1038/nrd2397
  2. Aranda, A. and A. Pascual. 2001. Nuclear hormone receptors and gene expression. Physiol. Rev. 81: 1269-1304.
  3. Bourguet, W., M. Ruff, P. Chambon, H. Gronemeyer, and D. Moras. 1995. Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-$\alpha$. Nature 375: 377-382. https://doi.org/10.1038/375377a0
  4. Dagher, R., C. Pigault, D. Bonnet, D. Boeglin, C. Pourbaix, M. C. Kilhoffer, et al. 2006. Use of a fluorescence polarization based high throughput assay to identify new calmodulin ligands. Biochim. Biophys. Acta 1763: 1250-1255. https://doi.org/10.1016/j.bbamcr.2006.09.027
  5. Fuchs, M. 2012. Non-alcoholic fatty liver disease: The bile acid-activated farnesoid X receptor as an emerging treatment target. J. Lipids DOI:10.1155/2012/934396.
  6. Gadaleta, R. M., K. J. van Erpecum, B. Oldenburg, E. C. Willemsen, W. Renooij, S. Murzilli, et al. 2011. Farnesoid X receptor activation inhibits inflammation and preserves the intestinal barrier in inflammatory bowel disease. Gut 60: 463-472. https://doi.org/10.1136/gut.2010.212159
  7. Hollman, D. A. A., A. Miloan, K. J. van Erpecum, and S. W. van Mil. 2012. Anti-inflammatory and metabolic actions of FXR: Insights into molecular mechanism. Biochem. Biophys. Acta 1821: 1443-1452.
  8. Jonker, J. W., C. Liddle, and M. Downes. 2012. FXR and PXR: Potential therapeutic targets in cholestasis. J. Steroid Biochem. Mol. Biol. 130: 147-158. https://doi.org/10.1016/j.jsbmb.2011.06.012
  9. Lafebvre, P., B. Cariou, F. Lien, F. Kuipers, and B. Staels. 2009. Role of bile acids and bile acid receptors in metabolic regulation. Physiol. Rev. 89: 147. https://doi.org/10.1152/physrev.00010.2008
  10. Laffitte, B. A., H. R. Kast, C. M. Nguyen, A. M. Zavacki, D. D. Moore, and P. A. Edwards. 2000. Identification of the DNA binding specificity and potential target genes for the farnesoid X-activated receptor. J. Biol. Chem. 275: 10638-10647. https://doi.org/10.1074/jbc.275.14.10638
  11. Lee, K. C. and W. L. Kraus. 2001. Nuclear receptors, coactivators and chromatin: New approaches, new insights. Trends Endocrinol. Metab. 12: 191-197. https://doi.org/10.1016/S1043-2760(01)00392-7
  12. Levy-Bimbot, M., G. Major, D. Courilleau, J. P. Blondeau, and Y. Levi. 2012. Tetrabromobisphenol-A disrupts thyroid hormone receptor A function in vitro: Use of fluorescence polarization to assay corepressor and coactivator peptide binding. Chemosphere 87: 782-788. https://doi.org/10.1016/j.chemosphere.2011.12.080
  13. Li, C., J. W. Schwabe, E. Banayo, and R. M. Evans. 1997. Coexpression of nuclear receptor partners increases their solubility and biological activities. Proc. Natl. Acad. Sci. USA 94: 2278-2283. https://doi.org/10.1073/pnas.94.6.2278
  14. Lu, T. T., M. Makishima, J. J. Repa, K. Schoonjans, T. A. Kerr, J. Auwerx, and D. J. Mangelsdorf. 2000. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Mol. Cell 6: 507-515. https://doi.org/10.1016/S1097-2765(00)00050-2
  15. Makishima, M., A. Y. Okamoto, J. J. Repa, H. Tu, R. M. Leaned, A. Luk, et al. 1999. Identification of a nuclear receptor for bile acids. Science 284: 1362-1365. https://doi.org/10.1126/science.284.5418.1362
  16. Mangelsdorf, D. J. and R. M. Evans. 1995. The RXR heterodimers and orphan receptor. Cell 83: 841-850. https://doi.org/10.1016/0092-8674(95)90200-7
  17. Mangelsdorf, D. J., C. Thummel, M. Beato, P. Herrich, G. Schultz, K. Umesono, et al. 1995. The nuclear receptor superfamily: The second decade. Cell 83: 835-839. https://doi.org/10.1016/0092-8674(95)90199-X
  18. McKenna, N. J., R. B. Lanz, and B. W. O'Malley. 1999. Nuclear receptor coregulators: Cellular and molecular biology. Endocrinol. Rev. 20: 321-344.
  19. Nagy, L. and J. W. R. Schwabe. 2004. Mechanism of the nuclear receptor molecular switch. Trends Biochem. Sci. 29: 317-324. https://doi.org/10.1016/j.tibs.2004.04.006
  20. Neuschwander-Tetri, B. A. 2012. Farnesoid X receptor agonists: What they are and how they might be used in treating liver disease. Curr. Gastroenterol. Rep. 14: 55-62. https://doi.org/10.1007/s11894-011-0232-6
  21. Ogawa, S., J. Lozach, C. Benner, G. Pascual, R. K. Tangirala, S. Westin, et al. 2005. Molecular determinants of crosstalk between nuclear receptors and toll-like receptors. Cell 122: 707-721. https://doi.org/10.1016/j.cell.2005.06.029
  22. Overington, J. P. and B. Al-Lazikani. 2006. How many drug targets are there? Nat. Rev. Drug Discov. 5: 993-996. https://doi.org/10.1038/nrd2199
  23. Parks, D. J., S. G. Blanchard, R. K. Bledsoe, G. Chandra, T. G. Consler, S. A. Kliewer, et al. 1999. Bile acids: Natural ligands for orphan nuclear receptor. Science 284: 1365-1358. https://doi.org/10.1126/science.284.5418.1365
  24. Saïda, F., M. Uzan, B. Odaert, and F. Bontems. 2006. Expression of highly toxic genes in E. coli: Special strategies and genetic tools. Curr. Protein Pept. Sci. 7: 47-56. https://doi.org/10.2174/138920306775474095
  25. Seol, W., H. S. Choi, and D. D. Moore. 1995. Isolation of proteins that interact specifically with the retinoid X receptor: Two novel orphan receptors. Mol. Endocrinol. 9: 72-85.
  26. Sørensen, H. P. and K. K. Mortensen. 2005. Advanced genetic strategies for recombinant protein expression in Escherichia coli. J. Biotechnol. 115: 113-128. https://doi.org/10.1016/j.jbiotec.2004.08.004
  27. Teodoro, J. S., A. P. Rolo, and C. M. Plameira. 2011. Hepatic FXR: Key regulator of whole-body energy metabolism. Trends Endocrinol. Metab. 22: 458-466. https://doi.org/10.1016/j.tem.2011.07.002
  28. Thomas, A. M., S. N. Hart, B. Kong, J. Fang, X. B. Zhong, and G. L. Guo. 2010. Genome-wide tissue-specific farnesoid X receptor binding in mouse liver and intestine. Hepatology 51: 1410-1419. https://doi.org/10.1002/hep.23450
  29. Vavassori, P., A. Mencarelli, B. Renga, E. Distrutti, and S. Fiorucci. 2009. The bile acid receptor FXR is a modulator of intestinal innate immunity. J. Immunol. 183: 6251-6261. https://doi.org/10.4049/jimmunol.0803978
  30. Wurtz, J. M., W. Bourguet, J. P. Renaud, V. Vivat, P. Chambon, D. Moras, and H. Gronemeyer. 1996. A canonical structure for the ligand-binding domain of nuclear receptors. Nat. Struct. Mol. Biol. 3: 87-94. https://doi.org/10.1038/nsb0196-87