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. 2013 Jan 17;28(1):43–48. doi: 10.1007/s12250-013-3286-9

Modification in media composition to obtain secretory production of STxB-based vaccines using Escherichia coli

Mohammad Sadraeian 1, Mohammad Bagher Ghoshoon 1,2, Milad Mohkam 1,2, Zeinab Karimi 1,2,3, Sara Rasoul-Amini 1,2,3, Younes Ghasemi 1,2,✉
PMCID: PMC8208459  PMID: 23329470

Abstract

Shiga toxin B-subunit (STxB) from Shigella dysenteriae targets in vivo antigen to cancer cells, dendritic cells (DC) and B cells, which preferentially express the globotriaosylceramide (Gb3) receptor. This pivotal role has encouraged scientists to investigate fusing STxB with other clinical antigens. Due to the challenges of obtaining a functional soluble form of the recombinant STxB, such as formation of inclusion bodies during protein expression, scientists tend to combine STxB with vaccine candidates rather than using their genetically fused forms. In this work, we fused HPV16 E7 as a vaccine candidate to the recombinantly-produced STxB. To minimize the formation of inclusion bodies, we investigated a number of conditions during the expression procedure. Then various strategies were used in order to obtain high yield of soluble recombinant protein from E. coli which included the use of different host strains, reduction of cultivation temperature, as well as using different concentrations of IPTG and different additives (Glycin, Triton X-100, ZnCl2). Our study demonstrated the importance of optimizing incubation parameters for recombinant protein expression in E. coli; also showed that the secretion production can be achieved over the course of a few hours when using additives such as glycine and Triton X-100. Interestingly, it was shown that when the culture mediums were supplemented by additives, there was an inverse ratio between time of induction (TOI) and the level of secreted protein at lower temperatures. This study determines the optimal conditions for high yield soluble E7-STxB expression and subsequently facilitates reaching a functionally soluble form of STxB-based vaccines, which can be considered as a potent vaccine candidate for cervical cancer.

Keywords: Protein vaccine, STxB, Soluble expression, Triton X-100

Footnotes

Foundation item: This work was supported by Research Council of Shiraz University of Medical Sciences (91-01-36-4417), Shiraz, Iran.

References

  1. Baneyx F., Mujacic M. Recombinant protein folding and misfolding inEscherichia coli. Nat Biotechnol. 2004;22:1399–1408. doi: 10.1038/nbt1029. [DOI] [PubMed] [Google Scholar]
  2. Buchner J., Pastan I., Brinkmann U. A method for increasing the yield of properly folded recombinant fusion proteins: single-chain immunotoxins from renaturation of bacterial inclusion bodies. Anal Biochem. 1992;205:263–270. doi: 10.1016/0003-2697(92)90433-8. [DOI] [PubMed] [Google Scholar]
  3. Choi J. H., Lee S. Y. Secretory and extracellular production of recombinant proteins usingEscherichia coli. Appl Microbiol Biotechnol. 2004;64:625–635. doi: 10.1007/s00253-004-1559-9. [DOI] [PubMed] [Google Scholar]
  4. Choi N. W., Estes M. K., Langridge W. H. R. Oral immunization with a shiga toxin B subunit: Rotavirus NSP4(90) fusion protein protects mice against gastroenteritis. Vaccine. 2005;23:5168–5176. doi: 10.1016/j.vaccine.2005.06.015. [DOI] [PubMed] [Google Scholar]
  5. Goncalves A. N., Meschiari C. A., Stetler-Stevenson W. G., Nonato M. C., Alves C. P., Espreafico E. M., Gerlach R. F. Expression of soluble and functional full-length human Matrix Metalloproteinase-2 inEscherichia coli. J Biotechnol. 2012;157:20–24. doi: 10.1016/j.jbiotec.2011.09.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Haicheur N., Bismuth E., Bosset S., Adotevi O., Warnier G., Lacabanne V., Regnault A., Desaymard C., Amigorena S., Ricciardi-Castagnoli P. The B subunit of Shiga toxin fused to a tumor antigen elicits CTL and targets dendritic cells to allow MHC class I-restricted presentation of peptides derived from exogenous antigens. J Immunol. 2000;165:3301–3309. doi: 10.4049/jimmunol.165.6.3301. [DOI] [PubMed] [Google Scholar]
  7. Haicheur N., Benchetrit F., Amessou M., Leclerc C., Falguires T., Fayolle C., Bismuth E., Fridman W. H., Johannes L., Tartour E. The B subunit of Shiga toxin coupled to full-size antigenic protein elicits humoral and cell-mediated immune responses associated with a Th1 dominant polarization. Int immunol. 2003;15:1161–1171. doi: 10.1093/intimm/dxg118. [DOI] [PubMed] [Google Scholar]
  8. Han L., Doverskog M., Enfors S. O., Häggström L. Effect of glycine on the cell yield and growth rate ofEscherichia coli: evidence for cell-density-dependent glycine degradation as determined by 13C NMR spectroscopy. J Biotechnol. 2002;92:237–249. doi: 10.1016/S0168-1656(01)00373-X. [DOI] [PubMed] [Google Scholar]
  9. Humphreys D. P., Sehdev M., Chapman A. P., Ganesh R., Smith B. J., King L. M., Glover D. J., Reeks D. G., Stephens P. E. High-level periplasmic expression inEscherichia coliusing a eukaryotic signal peptide: importance of codon usage at the 5′end of the coding sequence. Protein Expr Purif. 2000;20:252–264. doi: 10.1006/prep.2000.1286. [DOI] [PubMed] [Google Scholar]
  10. Janssen K. P., Vignjevic D., Boisgard R., Falguires T., Bousquet G., Decaudin D., Dolli F., Louvard D., Tavitian B., Robine S. In vivo tumor targeting using a novel intestinal pathogen-based delivery approach. Cancer Res. 2006;66:7230–7237. doi: 10.1158/0008-5472.CAN-06-0631. [DOI] [PubMed] [Google Scholar]
  11. LaCasse E. C., Saleh M. T., Patterson B., Minden M. D., Gariepy J. Shiga-like toxin purges human lymphoma from bone marrow of severe combined immunodeficient mice. Blood. 1996;88:1561–1567. [PubMed] [Google Scholar]
  12. Lee R. S., Tartour E., Van der Bruggen P., Vantomme V., Joyeux I., Goud B., Fridman W. H., Johannes L. Major histocompatibility complex class I presentation of exogenous soluble tumor antigen fused to the B-fragment of Shiga toxin. Eur J Immunol. 1998;28:2726–2737. doi: 10.1002/(SICI)1521-4141(199809)28:09<2726::AID-IMMU2726>3.0.CO;2-W. [DOI] [PubMed] [Google Scholar]
  13. Li J., Liu J., Lan X., Cheng J., Wu R., Lou Z. Z., Yin X., Li X., Li B., Yang B. Cloning the structure genes and expression the N gene of porcine epidemic diarrhea virus DX. Virol Sin. 2009;24:179–186. doi: 10.1007/s12250-009-2982-y. [DOI] [Google Scholar]
  14. Li Y. L., Liu J., Liu J. N., Zhang J. Immunization of protein HPV16 E7 in fusion with mouse HSP70 inhibits the growth of TC-1 cells in tumor bearing mice. Vaccine. 2011;29:5959–5962. doi: 10.1016/j.vaccine.2011.06.046. [DOI] [PubMed] [Google Scholar]
  15. Nakagawa I., Nakata M., Kawabata S., Hamada S. Regulated expression of the Shiga toxin B gene induces apoptosis in mammalian fibroblastic cells. Mol Microbiol. 1999;33:1190–1199. doi: 10.1046/j.1365-2958.1999.01564.x. [DOI] [PubMed] [Google Scholar]
  16. Ni Y., Chen R. Extracellular recombinant protein production fromEscherichia coli. Biotechnol Lett. 2009;31:1661–1670. doi: 10.1007/s10529-009-0077-3. [DOI] [PubMed] [Google Scholar]
  17. O’Brien A. D., Tesh V. L., Donohue-Rolfe A., Jackson M. P., Olsnes S., Sandvig K., Lindberg A. A., Keusch G. T. Shiga toxin: biochemistry, genetics, mode of action and role in pathogenesis. Curr Top Microbiol Immunol. 1992;180:65–94. doi: 10.1007/978-3-642-77238-2_4. [DOI] [PubMed] [Google Scholar]
  18. Ohmura M., Yamamoto M., Tomiyama-Miyaji C., Yuki Y., Takeda Y., Kiyono H. Nontoxic Shiga toxin derivatives fromEscherichia colipossess adjuvant activity for the augmentation of antigen-specific immune responses via dendritic cell activation. Infect Immu. 2005;73:4088–4097. doi: 10.1128/IAI.73.7.4088-4097.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sadraeian M., Honari H., Madanchi H., Hesaraki M. Cloning and Expression of CtxB-StxB inEsherichia coli: A challenge for Improvement of Immune Response Against StxB. Iran J Pharm Sci. 2011;7:185–190. [Google Scholar]
  20. Sedighzadeh S S, Shamsara M and Shahpiri A. 2012. Fusion Protein Strategy to Increase Expression and Solubility of Hypervariable Region of VP2 Protein of Infectious Bursal Disease Virus in Escherichia coli. Prot J: 1–5. [DOI] [PubMed]
  21. Shaw C. A., Starnbach M. N. Using modified bacterial toxins to deliver vaccine antigens. ASM News. 2003;69:384–389. [Google Scholar]
  22. Vingert B., Adotevi O., Patin D., Jung S., Shrikant P., Freyburger L., Eppolito C., Sapoznikov A., Amessou M., Quintin-Colonna F. The Shiga toxin B-subunit targets antigen in vivo to dendritic cells and elicits anti-tumor immunity. Eur J Immunol. 2006;36:1124–1135. doi: 10.1002/eji.200535443. [DOI] [PubMed] [Google Scholar]
  23. Wang P., Song P., Li X., Su R. R., Wang H., Zhu G. P. Study on soluble expression of glutamate dehydrogenase from tea plant in Escherichia coli using fusion tags. African J Biotechnol. 2012;11:6241–6250. [Google Scholar]
  24. Weikert C., Sauer U., Bailey J. E. An Escherichia coli host strain useful for efficient overproduction of secreted recombinant protein. Biotechnol Bioeng. 1998;59:386–391. doi: 10.1002/(SICI)1097-0290(19980805)59:3<386::AID-BIT16>3.0.CO;2-C. [DOI] [PubMed] [Google Scholar]
  25. Yang J., Moyana T., MacKenzie S., Xia Q., Xiang J. One Hundred Seventy-Fold Increase in Excretion of an FV Fragment-Tumor Necrosis Factor Alpha Fusion Protein (sFV/TNF-α) fromEscherichia coliCaused by the Synergistic Effects of Glycine and Triton X-100. Appl Environ Microbiol. 1998;64:2869–2874. doi: 10.1128/aem.64.8.2869-2874.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Zhang S., Fan R., Yang T., Sun Y., Li J., Xu Q., Zhou P. An improved strategy for efficient expression and purification of soluble HIV-1 tat protein inE. coli. Virol Sin. 2009;24:518–528. doi: 10.1007/s12250-009-3068-6. [DOI] [Google Scholar]

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