Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Novel Modification of Growth Medium Enables Efficient E. coli Expression and Simple Purification of an Endotoxin-Free Recombinant Murine Hsp70 Protein

  • Zachova, Katerinat (Department of Immunology, Faculty of Medicine and Dentistry, Palacky University in Olomouc) ;
  • Krupka, Michal (Department of Immunology, Faculty of Medicine and Dentistry, Palacky University in Olomouc) ;
  • Chamrad, Ivo (Department of Biochemistry, Faculty of Science, Palacky University in Olomouc) ;
  • Belakova, Jana (Department of Immunology, Faculty of Medicine and Dentistry, Palacky University in Olomouc) ;
  • Horynova, Milada (Department of Immunology, Faculty of Medicine and Dentistry, Palacky University in Olomouc) ;
  • Weigl, Evzen (Department of Immunology, Faculty of Medicine and Dentistry, Palacky University in Olomouc) ;
  • Sebela, Marek (Department of Biochemistry, Faculty of Science, Palacky University in Olomouc) ;
  • Raska, Milan (Department of Immunology, Faculty of Medicine and Dentistry, Palacky University in Olomouc)
  • Published : 2009.07.31
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Abstract

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-n-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite,N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other Histagged proteins expressed in E. coli.

Keywords

References

  1. Aida, Y. and M. J. Pabst. 1990. Removal of endotoxin from protein solutions by phase separation using Triton X-114. J. Immunol. Methods 132: 191-195 https://doi.org/10.1016/0022-1759(90)90029-U
  2. Bausinger, H., D. Lipsker, U. Ziylan, S. Manie, J. P. Briand, J. P. Cazenave, et al. 2002. Endotoxin-free heat-shock protein 70 fails to induce APC activation. Eur. J. Immunol 32: 3708-3713 https://doi.org/10.1002/1521-4141(200212)32:12<3708::AID-IMMU3708>3.0.CO;2-C
  3. Baykov, A. A., O. A. Evtushenko, and S. M. Avaeva. 1988. A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay. Anal. Biochem. 171: 266-270 https://doi.org/10.1016/0003-2697(88)90484-8
  4. Benaroudj, N., B. Fang, F. Triniolles, C. Ghelis, and M. M. Ladjimi. 1994. Overexpression in Escherichia coli, purification and characterization of the molecular chaperone HSC70. Eur. J. Biochem. 221: 121-128 https://doi.org/10.1111/j.1432-1033.1994.tb18720.x
  5. Geladopoulos, T. P., T. G. Sotiroudis, and A. E. Evangelopoulos. 1991. A malachite green colorimetric assay for protein phosphatase activity. Anal. Biochem. 192: 112-116 https://doi.org/10.1016/0003-2697(91)90194-X
  6. Hauser, H. and S. Y. Chen. 2003. Augmentation of DNA vaccine potency through secretory heat shock protein-mediated antigen targeting. Methods 31: 225-231 https://doi.org/10.1016/S1046-2023(03)00136-1
  7. Hauser, H., L. Shen, Q. L. Gu, S. Krueger, and S. Y. Chen. 2004. Secretory heat-shock protein as a dendritic cell-targeting molecule: A new strategy to enhance the potency of genetic vaccines. Gene Ther. 11: 924-932 https://doi.org/10.1038/sj.gt.3302160
  8. Lipsker, D., U. Ziylan, D. Spehner, F. Proamer, H. Bausinger, P. Jeannin, et al. 2002. Heat shock proteins 70 and 60 share common receptors which are expressed on human monocyte-derived but not epidermal dendritic cells. Eur. J. Immunol. 32: 322-332 https://doi.org/10.1002/1521-4141(200202)32:2<322::AID-IMMU322>3.0.CO;2-0
  9. Malyala, P. and M. Singh. 2008. Endotoxin limits in formulations for preclinical research. J. Pharm. Sci. 97: 2041-2044 https://doi.org/10.1002/jps.21152
  10. Menoret, A. 2004. Purification of recombinant and endogenous HSP70s. Methods 32: 7-12 https://doi.org/10.1016/S1046-2023(03)00180-4
  11. Nadeau, K., A. Das, and C. T. Walsh. 1993. Hsp90 chaperonins possess ATPase activity and bind heat shock transcription factors and peptidyl prolyl isomerases. J. Biol. Chem. 268: 1479-1487
  12. Nadeau, K., M. A. Sullivan, M. Bradley, D. M. Engman, and C. T. Walsh. 1992. 83-Kilodalton heat shock proteins of trypanosomes are potent peptide-stimulated ATPases. Protein Sci. 1: 970-979 https://doi.org/10.1002/pro.5560010802
  13. Olson, C. L., K. C. Nadeau, M. A. Sullivan, A. G. Winquist, J. E. Donelson, C. T. Walsh, and D. M. Engman. 1994. Molecular and biochemical comparison of the 70-kDa heat shock proteins of Trypanosoma cruzi. J. Biol. Chem. 269: 3868-3874
  14. Raska, M., J. Belakova, M. Horynova, M. Krupka, J. Novotny, M. Sebestova, and E. Weigl. 2008. Systemic and mucosal immunization with Candida albicans hsp90 elicits hsp90-specific humoral response in vaginal mucosa which is further enhanced during experimental vaginal candidiasis. Med. Mycol. 46: 411-420 https://doi.org/10.1080/13693780701883508
  15. Raska, M., J. Belakova, N. K. Wudattu, L. Kafkova, K. Ruzickova, M. Sebestova, Z. Kolar, and E. Weigl. 2005. Comparison of protective effect of protein and DNA vaccines hsp90 in murine model of systemic candidiasis. Folia Microbiol. (Praha) 50: 77-82 https://doi.org/10.1007/BF02931297
  16. Sebela, M., T. Stosova, J. Havlis, N. Wielsch, H. Thomas, Z. Zdrahal, and A. Shevchenko. 2006. Thermostable trypsin conjugates for high-throughput proteomics: Synthesis and performance evaluation. Proteomics 6: 2959-2963 https://doi.org/10.1002/pmic.200500576
  17. Stosova, T., M. Sebela, P. Rehulka, O. Sedo, J. Havlis, and Z. Zdrahal. 2008. Evaluation of the possible proteomic application of trypsin from Streptomyces griseus. Anal. Biochem. 376: 94-102 https://doi.org/10.1016/j.ab.2008.01.016
  18. Suzue, K., X. Zhou, H. N. Eisen, and R. A. Young. 1997. Heat shock fusion proteins as vehicles for antigen delivery into the major histocompatibility complex class I presentation pathway. Proc. Natl. Acad. Sci. U.S.A. 94: 13146-13151 https://doi.org/10.1073/pnas.94.24.13146
  19. Udono, H., T. Saito, M. Ogawa, and Y. Yui. 2004. Hsp-antigen fusion and their use for immunization. Methods 32: 21-24 https://doi.org/10.1016/S1046-2023(03)00182-8
  20. Valentinis, B., A. Capobianco, F. Esposito, A. Bianchi, P. Rovere-Querini, A. A. Manfredi, and C. Traversari. 2008. Human recombinant heat shock protein 70 affects the maturation pathways of dendritic cells in vitro and has an in vivo adjuvant activity. J. Leukoc. Biol. 84: 199-206 https://doi.org/10.1189/jlb.0807548
  21. Vanhove, M., S. Houba, J. b1motte-Brasseur, and J. M. Frere. 1995. Probing the determinants of protein stability: Comparison of class A beta-lactamases. Biochem. J. 308(Pt 3): 859-864 https://doi.org/10.1042/bj3080859
  22. Waugh, D. F. 1954. Protein-protein interactions. Adv. Protein Chem. 9: 325-437 https://doi.org/10.1016/S0065-3233(08)60210-7
  23. Wieland, A., M. Denzel, E. Schmidt, S. Kochanek, F. Kreppel, J. Reimann, and R. Schirmbeck. 2008. Recombinant complexes of antigen with stress proteins are potent CD8 T-cell-stimulating immunogens. J. Mol. Med. 86: 1067-1079 https://doi.org/10.1007/s00109-008-0371-x
  24. Ye, Z. and Y. H. Gan. 2007. Flagellin contamination of recombinant heat shock protein 70 is responsible for its activity on T cells. J. Biol. Chem. 282: 4479-4484 https://doi.org/10.1074/jbc.M606802200

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