Skip to main content
SpringerLink
Log in

Development of Heterologous Expression System and Optimization of the Method of Cholera Toxin β-Subunit Production in E. coli

  • Published:
Biochemistry (Moscow) Aims and scope Submit manuscript

Abstract

Cholera is a deadly infection disease, which is usually associated with low hygiene levels and limited access to high-quality drinking water. An effective way to prevent cholera is the use of vaccines. Among active vaccine components there is the CtxB protein (cholera toxin β-subunit). In the current work, we have developed a genetic system for production of the recombinant CtxB in E. coli cells and studied conditions for synthesis and purification of the target product at the laboratory scale. It has been found that the optimal algorithm for isolation of the recombinant protein is to grow E. coli culture in the synthetic M9 medium with glycerol, followed by CtxB purification out of the spent culture medium using Ni2+-chelate affinity chromatography techniques. Forty-eight hours after induction of CtxB expression, concentration of the target product could be up to 50 mg/liter in the culture medium. The CtxB protein retains its pentameric structure during expression and through purification. The latter makes it possible to consider the developed system as a promising tool for the industrial-level production of recombinant CtxB for medical and research purposes.

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

Access this article

Log in via an institution

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

Abbreviations

CtxB:

β-subunit of cholera toxin

IPTG:

isopropyl β-d-1-thiogalactopyranoside

MALDI-TOF:

matrix-assisted laser desorption ionization – time of flight mass spectrometry

NB:

Nutrient Broth, complete nutrient medium similar in composition to Luria Bertani broth (LB)

References

  1. Almagro-Moreno, S., and Taylor, R. K. (2013) Cholera: environmental reservoirs and impact on disease transmission, Microbiol. Spectr., 1, https://doi.org/10.1128/microbiolspec.OH-0003-2012.

    Article  PubMed  Google Scholar 

  2. Hu, D., Liu, B., Feng, L., Ding, P., Guo, X., Wang, M., Cao, B., Reeves, P. R., and Wang, L. (2016) Origins of the current seventh cholera pandemic, Proc. Natl. Acad. Sci. USA, 113, E7730-E7739, https://doi.org/10.1073/pnas.1608732113.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. WHO, TheWeekly Epidemiological Record (2021) URL: https://www.who.int/publications/journals/weeklyepidemiological-record (accessed on 18.08.2023).

  4. WHO, https://www.who.int/emergencies/disease-outbreak-news/item/2023-DON437 (accessed on 18.08.2023).

  5. Clemens, J., Shin, S., Sur, D., Nair, G. B., and Holmgren, J. (2011) New-generation vaccines against cholera, Nat. Rev. Gastroenterol. Hepatol., 8, 701-710, https://doi.org/10.1038/nrgastro.2011.174.

    Article  CAS  PubMed  Google Scholar 

  6. Hui Xian, T., Parasuraman, S., Ravichandran, M., and Prabhakaran, G. (2022) Dual-use vaccine for diarrhoeal diseases: cross-protective immunogenicity of a cold-chain-free, live-attenuated, oral cholera vaccine against enterotoxigenic Escherichia coli (ETEC) challenge in BALB/c mice, Vaccines, 10, 2161, https://doi.org/10.3390/vaccines10122161.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. WHO. Cholera vaccine, URL: https://www.who.int/teams/immunization-vaccines-and-biologicals/diseases/cholera (accessed on 18.08.2023).

  8. Ravichandran, M., Tew, H. X., Prabhakaran, G., Parasuraman, S., and Norazmi, M. N. (2022) Live, genetically attenuated, cold-chain-free cholera vaccine – a research and development journey: light at the end of a long tunnel, Malays. J. Med. Sci., 29, 1-7, https://doi.org/10.21315/mjms2022.29.2.1.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Sumarokov, A. A., Ivanov, N. R., Dzhaparidze, M. N., Rystsova, E. A., Reznikov, Iu. B., et al. (1991) The characteristics of the reactogenicity and immunological activity of a new cholera bivalent chemical vaccine based on the results of controlled trials, Zhurn. Mikrobiol. Epidemiol. Immunobiol., 7, 55-58.

    Google Scholar 

  10. Sumarokov, A. A., Dzhaparidze, M. N., Eliseev, Iu. Iu., Nikitina, G. P., Poliakov, K. A., et al. (1993) The determination of the optimal inoculation dose of an oral cholera bivalent chemical vaccine in a controlled trial of the vaccination of children and adolescents, Zhurn. Mikrobiol. Epidemiol. Immunobiol., 5, 55-60.

    Google Scholar 

  11. Cholera vaccine bivalent, chemical. User manual, URL: https://www.vidal.ru/drugs/cholera_bivalent_chemical_vaccine__42897 (accessed on 18.08.2023).

  12. Zhang, R. G., Westbrook, M. L., Westbrook, E. M., Scott, D. L., and Otwinowski, Z. (1995) The 2.4 A crystal structure of cholera toxin B subunit pentamer: choleragenoid, J. Mol. Biol., 251, 550-562, https://doi.org/10.1006/jmbi.1995.0455.

    Article  CAS  PubMed  Google Scholar 

  13. Korotkov, K. V., Sandkvist, M., and Hol, W. G. (2012) The type II secretion system: biogenesis, molecular architecture and mechanism, Nat. Rev. Microbiol., 10, 336-351, https://doi.org/10.1038/nrmicro2762.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Overbye, L. J., Sandkvist, M., and Bagdasarian, M. (1993) Genes required for extracellular secretion of enterotoxin are clustered in Vibrio cholerae, Gene, 132, 101-106, https://doi.org/10.1016/0378-1119(93)90520-D.

    Article  CAS  PubMed  Google Scholar 

  15. Wernick, N. L., Chinnapen, D. J., Cho, J. A., and Lencer, W. I. (2010) Cholera toxin: an intracellular journey into the cytosol by way of the endoplasmic reticulum, Toxins (Basel), 2, 310-325, https://doi.org/10.3390/toxins2030310.

    Article  CAS  PubMed  Google Scholar 

  16. Baldauf, K. J., Royal, J. M., Hamorsky, K. T., and Matoba, N. (2015) Cholera toxin B: one subunit with many pharmaceutical applications., Toxins (Basel), 7, 974-996, https://doi.org/10.3390/toxins7030974.

    Article  CAS  PubMed  Google Scholar 

  17. Jelinek, T., and Kollaritsch, H. (2008) Vaccination with Dukoral against travelers’ diarrhea (ETEC) and cholera, Expert Rev. Vaccines, 7, 561-567, https://doi.org/10.1586/14760584.7.5.561.

    Article  CAS  PubMed  Google Scholar 

  18. Lebens, M., Johansson, S., Osek, J., Lindblad, M., and Holmgren, J. (1993) Large-scale production of Vibrio cholerae toxin B subunit for use in oral vaccines, Biotechnology, 11, 1574-1578, https://doi.org/10.1038/nbt1293-1574.

    Article  CAS  PubMed  Google Scholar 

  19. Rhie, G. E., Jung, H. M., Park, J., Kim, B. S., and Mekalanos, J. J. (2008) Construction of cholera toxin B subunit-producing Vibrio cholerae strains using the Mariner-FRT transposon delivery system, FEMS Immunol. Med. Microbiol., 52, 23-28, https://doi.org/10.1111/j.1574-695X.2007.00346.x.

    Article  CAS  PubMed  Google Scholar 

  20. Slos, P., Dutot, P., Reymund, J., Kleinpeter, P., and Prozzi, D. (1998) Production of cholera toxin B subunit in Lactobacillus, FEMS Microbiol. Lett., 169, 29-36, https://doi.org/10.1111/j.1574-6968.1998.tb13295.x.

    Article  CAS  PubMed  Google Scholar 

  21. Goto, N., Maeyama, J., Yasuda, Y., Isaka, M., and Matano, K. (2000) Safety evaluation of recombinant cholera toxin B subunit produced by Bacillus brevis as a mucosal adjuvant, Vaccine, 18, 2164-2171, https://doi.org/10.1016/s0264-410x(99)00337-0.

    Article  CAS  PubMed  Google Scholar 

  22. Zeighami, H., Sattari, M., and Rezayat, M. (2010) Cloning and expression of a cholera toxin beta subunit in Escherichia coli, Ann. Microbiol., 60, 451-454, https://doi.org/10.1007/s13213-010-0062-z.

    Article  CAS  Google Scholar 

  23. Dakterzada, F., Mobarez, A. M., Roudkenar, M. H., and Forouzandeh, M. (2012) Production of pentameric cholera toxin B subunit in Escherichia coli, Avicenna J. Med. Biotechnol., 4, 89-94.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Slos, P., Speck, D., Accart, N., Kolbe, H. V., and Schubnel, D. (1994) Recombinant cholera toxin B subunit in Escherichia coli: high-level secretion, purification, and characterization, Protein Expr. Purif., 5, 518-526, https://doi.org/10.1006/prep.1994.1071.

    Article  CAS  PubMed  Google Scholar 

  25. Matoba, N. (2015) N-glycosylation of CHOLERA toxin B subunit: serendipity for novel plant-made vaccines? Front Plant Sci., 6, 1132, https://doi.org/10.3389/fpls.2015.01132.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Hamorsky, K. T., Kouokam, J. C., Bennett, L. J., Baldauf, K. J., and Kajiura, H. (2013) Rapid and scalable plant-based production of a cholera toxin B subunit variant to aid in mass vaccination against cholera outbreaks, PLoS Negl. Trop. Dis., 7, e2046, https://doi.org/10.1371/journal.pntd.0002046.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Mudrak, B., and Kuehn, M. J. (2010) Specificity of the type II secretion systems of enterotoxigenic Escherichia coli and Vibrio cholerae for heat-labile enterotoxin and cholera toxin, J. Bacteriol., 192, 1902-1911, https://doi.org/10.1128/JB.01542-09.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Strain – DH5α. The Coli Genetic Stock Center (CGSC). Yale University, URL: https://cgsc.biology.yale.edu/Strain.php?ID=150015 (accessed on 18.08.2023).

  29. Karpov, D. S., Goncharenko, A. V., Usachev, E. V., Vasina, D. V., Divisenko, E. V., Chalenko, Y. M., Pochtovyi, A. A., Ovchinnikov, R. S., Makarov, V. V., Yudin, S. M., Tkachuk, A. P., and Gushchin, V. A. (2021) A strategy for the rapid development of a safe Vibrio cholerae candidate vaccine strain, Int. J. Mol. Sci., 22, 11657, https://doi.org/10.3390/ijms222111657.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Quan, S., Hiniker, A., Collet, J. F., and Bardwell, J. C. (2013) Isolation of bacteria envelope proteins, Methods Mol. Biol., 966, 359-366, https://doi.org/10.1007/978-1-62703-245-2_22.

    Article  CAS  PubMed  Google Scholar 

  31. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227, 680-685, https://doi.org/10.1038/227680a0.

    Article  CAS  PubMed  Google Scholar 

  32. Brunelle, J. L., and Green, R. (2014) One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE), Methods Enzymol., 541, 151-159, https://doi.org/10.1016/B978-0-12-420119-4.00012-4.

    Article  CAS  PubMed  Google Scholar 

  33. GelAnalyzer 19.1, URL: http://www.gelanalyzer.com/ (accessed: 18.08.2023).

  34. Hamorsky, K., and Matoba, N. (2016) Facile method for the production of recombinant cholera toxin B subunit in E. coli, Methods Mol. Biol., 1404, 511-518, https://doi.org/10.1007/978-1-4939-3389-1_33.

    Article  PubMed  Google Scholar 

  35. Naha, A., Mandal, R. S., Samanta, P., Saha, R. N., and Shaw, S. (2020) Deciphering the possible role of ctxB7 allele on higher production of cholera toxin by Haitian variant Vibrio cholerae O1, PLoS Negl. Trop. Dis., 14, e0008128, https://doi.org/10.1371/journal.pntd.0008128.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Wang, Y. (2002) The function of OmpA in Escherichia coli, Biochem. Biophys. Res. Commun., 292, 396-401, https://doi.org/10.1006/bbrc.2002.6657.

    Article  CAS  PubMed  Google Scholar 

  37. Keen, N. T., and Tamaki, S. (1986) Structure of two pectate lyase genes from Erwinia chrysanthemi EC16 and their high-level expression in Escherichia coli, J. Bacteriol., 168, 595-606, https://doi.org/10.1128/jb.168.2.595-606.1986.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Lei, S. P., Lin, H. C., Wang, S. S., Callaway, J., and Wilcox, G. (1987) Characterization of the Erwinia carotovora pelB gene and its product pectate lyase, J. Bacteriol., 169, 4379-4383, https://doi.org/10.1128/jb.169.9.4379-4383.1987.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Ruppert, A., Arnold, N., and Hobom, G. (1994) OmpA-FMDV VP1 fusion proteins: production, cell surface exposure and immune responses to the major antigenic domain of foot-and-mouth disease virus, Vaccine, 12, 492-498, https://doi.org/10.1016/0264-410x(94)90305-0.

    Article  CAS  PubMed  Google Scholar 

  40. Low, K. O., Mahadi, M. N., and Illias, R. M. (2013) Optimisation of signal peptide for recombinant protein secretion in bacterial hosts, Appl. Microbiol. Biotechnol., 97, 3811-3826, https://doi.org/10.1007/s00253-013-4831-z.

    Article  CAS  PubMed  Google Scholar 

  41. Zhou, Y., Lu, Z., Wang, X., Selvaraj, J. N., and Zhang, G. (2018) Genetic engineering modification and fermentation optimization for extracellular production of recombinant proteins using Escherichia coli, Appl. Microbiol. Biotechnol., 102, 1545-1556, https://doi.org/10.1007/s00253-017-8700-z.

    Article  CAS  PubMed  Google Scholar 

  42. Lee, J. H., Choi, S. Y., Jeon, Y. S., Lee, H. R., and Kim, E. J. (2009) Classification of hybrid and altered Vibrio cholerae strains by CTX prophage and RS1 element structure, J. Microbiol., 47, 783-788, https://doi.org/10.1007/s12275-009-0292-6.

    Article  CAS  PubMed  Google Scholar 

  43. Dertzbaugh, M. T., and Cox, L. M. (1998) The affinity of cholera toxin for Ni2+ ion, Protein Eng., 11, 577-581, https://doi.org/10.1093/protein/11.7.577.

    Article  CAS  PubMed  Google Scholar 

  44. Zhu, P., Stanisheuski, S., Franklin, R., Vogel, A., Vesely, C. H., Reardon, P., Sluchanko, N. N., Beckman, J. S., Karplus, P. A., Mehl, R. A., and Cooley, R. B. (2023) Autonomous synthesis of functional, permanently phosphorylated proteins for defining the interactome of monomeric 14-3-3ζ, ACS Cent Sci., 9, 816-835, https://doi.org/10.1021/acscentsci.3c00191.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Shatov, V. M., Muranova, L. K., Zamotina, M. A., Sluchanko, N. N., and Gusev, N. B. (2023) α-Crystallin domains of five human small heat shock proteins (sHsps) differ in dimer stabilities and ability to incorporate themselves into oligomers of full-length sHsps, Int. J. Mol. Sci., 24, 1085, https://doi.org/10.3390/ijms24021085.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Slonimskiy, Y. B., Zupnik, A. O., Varfolomeeva, L. A., Boyko, K. M., Maksimov, E. G., and Sluchanko, N. N. (2022) A primordial orange carotenoid protein: structure, photoswitching activity and evolutionary aspects, Int. J. Biol. Macromol., 222, 167-180, https://doi.org/10.1016/j.ijbiomac.2022.09.131.

    Article  CAS  PubMed  Google Scholar 

  47. Ianishevsky, N. V., Gintsburg, A. L., Vertiev, Yu. V., Demme, Yu. M., and Karataev, G. I. (1987) Construction of recombinant plasmid encoding biosynthesis of β-subunits of cholera toxin, Mol. Genet., 4, 26-32.

    Google Scholar 

  48. Smirnova, N. I., Chekhovskaya, G. V., Livanova, L. F., and Kobkova, I. M. (2004) Escherichia coli KM 147 stain producer of β-subunit of cholera toxin, Patent Russian Federation No. 2238975.

Download references

Acknowledgments

The authors are grateful to the staff of the Center for Collective Use “Industrial Biotechnology”, Federal Research Center for Biotechnology, Russian Academy of Sciences, for their help in conducting MALDI-TOF-analysis.

Funding

This work was financially supported by the Russian Science Foundation, grant no. 23-74-30004.

Author information

Authors and Affiliations

  1. Bach Institute of Biochemistry, Federal Research Center for Biotechnology, Russian Academy of Sciences, 119071, Moscow, Russia

    Hamesd H. Jamgochian, Mikhail V. Zamakhaev, Nikolai N. Sluchanko, Anna V. Goncharenko & Mikhail S. Shumkov

Authors
  1. Hamesd H. Jamgochian
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mikhail V. Zamakhaev
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nikolai N. Sluchanko
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anna V. Goncharenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mikhail S. Shumkov
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.S.Sh., A.V.G., concept and supervision of the study; H.H.J., M.S.Sh., M.V.Z., N.N.S., conducting experiments; M.S.Sh., A.V.G., discussion of the results of the study; M.S.Sh., M.V.Z., writing text of the paper; M.V.Z., editing text of the paper.

Corresponding author

Correspondence to Anna V. Goncharenko.

Ethics declarations

The authors declare no conflict of interest in financial or any other sphere. This article does not describe any studies involving humans or animals performed by any of the authors.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jamgochian, H.H., Zamakhaev, M.V., Sluchanko, N.N. et al. Development of Heterologous Expression System and Optimization of the Method of Cholera Toxin β-Subunit Production in E. coli. Biochemistry Moscow 88, 1304–1317 (2023). https://doi.org/10.1134/S0006297923090109

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0006297923090109

Keywords

Search

Navigation