Skip to main content
Log in

A Novel Method for Efficient Preparation of Mucosal Adjuvant Escherichia coli Heat-Labile Enterotoxin Mutant (LTm) by Artificially Assisted Self-Assembly In Vitro

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

As well-known powerful mucosal adjuvant proteins, Escherichia coli heat-labile enterotoxin (LT) and its non-toxic or low-toxic mutants (LTm) are capable of promoting strong mucosal immune responses to co-administered antigens in various types of vaccines. However, due to the complex composition and special structure, the yield of LTm directly from the recombinant genetic engineering strains is quite low. Here, we put forward a novel method to prepare LTm protein which designed, expressed, and purified three kinds of component subunits respectively and assembled them into a hexamer structure in vitro by two combination modes. In addition, by simulated in vivo environment of polymer protein assembly, the factors of the protein solution system which include environment temperature, pH, ionic strength of the solution, and ratio between each subunit were taken into consideration. Finally, we confirmed the optimal conditions of two assembly strategies and prepared the hexamer holotoxin in vitro. These results are not only an important significance in promoting large-scale preparation of the mucosal adjuvant LTm but also an enlightening to produce other multi-subunit proteins.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Reference

  1. Jana, S., & Deb, J. K. (2005). Strategies for efficient production of heterologous proteins in Escherichia coli. Applied Microbiology and Biotechnology, 67, 289–298.

    Article  CAS  Google Scholar 

  2. Baneyx, F., & Mujacic, M. (2004). Recombinant protein folding and misfolding in Escherichia coli. Nature Biotechnology, 22, 1399–1408.

    Article  CAS  Google Scholar 

  3. Brondyk, W. H. (2009). Selecting an appropriate method for expressing a recombinant protein. Methods in Enzymology, 463, 131–147.

    Article  CAS  Google Scholar 

  4. Demain, A. L., & Vaishnav, P. (2009). Production of recombinant proteins by microbes and higher organisms. Biotechnology Advances, 27, 297–306.

    Article  CAS  Google Scholar 

  5. Gopal, G. J., & Kumar, A. (2013). Strategies for the production of recombinant protein in Escherichia coli. The Protein Journal, 32, 419–425.

    Article  CAS  Google Scholar 

  6. Hartl, F. U., Bracher, A., & Hayer-Hartl, M. (2011). Molecular chaperones in protein folding and proteostasis. Nature, 475, 324–332.

    Article  CAS  Google Scholar 

  7. Fernandez-Lafuente, R. (2009). Stabilization of multimeric enzymes: strategies to prevent subunit dissociation. Enzyme and Microbial Technology, 45, 405–418.

    Article  CAS  Google Scholar 

  8. Schlieker, C., Bukau, B., & Mogk, A. (2002). Prevention and reversion of protein aggregation by molecular chaperones in the E. coli cytosol: implications for their applicability in biotechnology. Journal of Biotechnology, 96, 13–21.

    Article  CAS  Google Scholar 

  9. Spangler, B. D. (1992). Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. Microbiological Reviews, 56, 622–647.

    CAS  Google Scholar 

  10. Merritt, E. A., Pronk, S. E., Sixma, T. K., Kalk, K. H., van Zanten, B. A., & Hol, W. G. (1994). Structure of partially-activated E. coli heat-labile enterotoxin (LT) at 2.6 A resolution. FEBS Letters, 337, 88–92.

    Article  CAS  Google Scholar 

  11. Rappuoli, R., Pizza, M., Douce, G., & Dougan, G. (1999). Structure and mucosal adjuvanticity of cholera and Escherichia coli heat-labile enterotoxins. Immunology Today, 20, 493–500.

    Article  CAS  Google Scholar 

  12. Norton, E. B., Lawson, L. B., Freytag, L. C., & Clements, J. D. (2011). Characterization of a mutant Escherichia coli heat-labile toxin, LT(R192G/L211A), as a safe and effective oral adjuvant. Clinical and Vaccine Immunology, 18, 546–551.

    Article  CAS  Google Scholar 

  13. El-Kamary, S. S., Cohen, M. B., Bourgeois, A. L., Van De Verg, L., Bauers, N., Reymann, M., Pasetti, M. F., & Chen, W. H. (2013). Safety and immunogenicity of a single oral dose of recombinant double mutant heat-labile toxin derived from enterotoxigenic Escherichia coli. Clinical and Vaccine Immunology, 20, 1764–1770.

    Article  CAS  Google Scholar 

  14. Sjokvist Ottsjo, L., Flach, C. F., Clements, J., Holmgren, J., & Raghavan, S. (2013). A double mutant heat-labile toxin from Escherichia coli, LT(R192G/L211A), is an effective mucosal adjuvant for vaccination against Helicobacter pylori infection. Infection and Immunity, 81, 1532–1540.

    Article  Google Scholar 

  15. da Hora, V. P., Conceicao, F. R., Dellagostin, O. A., & Doolan, D. L. (2011). Non-toxic derivatives of LT as potent adjuvants. Vaccine, 29, 1538–1544.

    Article  Google Scholar 

  16. Lasaro, M. A., Rodrigues, J. F., Mathias-Santos, C., Guth, B. E., Regua-Mangia, A., Piantino Ferreira, A. J., Takagi, M., Cabrera-Crespo, J., Sbrogio-Almeida, M. E., & de Souza Ferreira, L. C. (2006). Production and release of heat-labile toxin by wild-type human-derived enterotoxigenic Escherichia coli. FEMS Immunology and Medical Microbiology, 48, 123–131.

    Article  CAS  Google Scholar 

  17. Bordenave-Juchereau, S., Almeida, B., Piot, J.-M., & Sannier, F. (2005). Effect of protein concentration, pH, lactose content and pasteurization on thermal gelation of acid caprine whey protein concentrates. Journal of Dairy Research, 72, 34–38.

    Article  CAS  Google Scholar 

  18. Hianik, T., Ostatna, V., Sonlajtnerova, M., & Grman, I. (2007). Influence of ionic strength, pH and aptamer configuration for binding affinity to thrombin. Bioelectrochemistry, 70, 127–133.

    Article  CAS  Google Scholar 

  19. Huang, D. M., & Chandler, D. (2000). Temperature and length scale dependence of hydrophobic effects and their possible implications for protein folding. Proceedings of the National Academy of Sciences of the United States of America, 97, 8324–8327.

    Article  CAS  Google Scholar 

  20. Chen, X., Zaro, J. L., & Shen, W. C. (2013). Fusion protein linkers: property, design and functionality. Advanced Drug Delivery Reviews, 65, 1357–1369.

    Article  CAS  Google Scholar 

  21. Green, M. R., & Sambrook, J. (2012). Molecular cloning: a laboratory manual (Vol. 1). York: Cold Spring Harbor Laboratory Press New.

    Google Scholar 

  22. Laskowski, R. A., Rullmannn, J. A., MacArthur, M. W., Kaptein, R., & Thornton, J. M. (1996). AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. Journal of Biomolecular NMR, 8, 477–486.

    Article  CAS  Google Scholar 

  23. Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in Microbiology, 5, 172.

    Google Scholar 

  24. Brasch, H., Iven, H., & Körner, J. (1982). Significance of acid base status, respiration, Na+−and K+−concentrations and plasma glucose for acute toxicity of TRIS (hydroxymethyl-)aminomethane in rats. Archives of Toxicology, 51, 139–149.

    Article  CAS  Google Scholar 

  25. Talley, K., & Alexov, E. (2010). On the pH-optimum of activity and stability of proteins. Proteins, 78, 2699–2706.

    CAS  Google Scholar 

  26. Vera, A., Gonzalez-Montalban, N., Aris, A., & Villaverde, A. (2007). The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures. Biotechnology and Bioengineering, 96, 1101–1106.

    Article  CAS  Google Scholar 

  27. Takahashi, T. (1997). Significant role of electrostatic interactions for stabilization of protein assemblies. Advances in Biophysics, 34, 41–54.

    Article  CAS  Google Scholar 

  28. Kunkel, S. L., & Robertson, D. C. (1979). Purification and chemical characterization of the heat-labile enterotoxin produced by enterotoxigenic Escherichia coli. Infection and Immunity, 25, 586–596.

    CAS  Google Scholar 

  29. Yamamoto, T., Tamura, T., & Yokota, T. (1984). Primary structure of heat-labile enterotoxin produced by Escherichia coli pathogenic for humans. The Journal of Biological Chemistry, 259, 5037–5044.

    CAS  Google Scholar 

  30. Uesaka, Y., Otsuka, Y., Lin, Z., Yamasaki, S., Yamaoka, J., Kurazono, H., & Takeda, Y. (1994). Simple method of purification of Escherichia coli heat-labile enterotoxin and cholera toxin using immobilized galactose. Microbial Pathogenesis, 16, 71–76.

    Article  CAS  Google Scholar 

  31. Yamaguchi, H., & Miyazaki, M. (2014). Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies. Biomolecules, 4, 235–251.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This study was supported by the National Natural Science Foundation (30873190, 31300660), Science and Technology Innovation Action Plan of Shanghai (14431904300), Shanghai Pujiang Program (13PJD012), and a foundation for young teachers from Education Ministry of China (20120074120027) and partially supported by the Open Funding Project of the State Key Laboratory of Bioreactor Engineering.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Ping Wang or Xingyuan Ma.

Ethics declarations

Conflict of Interest

The authors declare no conflicts of interest. The authors are responsible for the content and writing of the paper.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liu, D., Zhang, N., Zheng, W. et al. A Novel Method for Efficient Preparation of Mucosal Adjuvant Escherichia coli Heat-Labile Enterotoxin Mutant (LTm) by Artificially Assisted Self-Assembly In Vitro. Appl Biochem Biotechnol 179, 33–45 (2016). https://doi.org/10.1007/s12010-015-1977-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-015-1977-4

Keywords

Navigation