Skip to main content

Advertisement

SpringerLink
Log in

Expression of Cholera Toxin B Subunit in Transgenic Rice Endosperm

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

The synthetic cholera toxin B subunit (CTB) gene, modified according to the optimized codon usage of plant genes, was introduced into a plant expression vector and expressed under the control of the Bx17 HMW (high molecular weight) wheat endosperm-specific promoter containing an intron of the rice act1. The recombinant vector was transformed into rice plants using a biolistic-mediated transformation method. Stable integration of the synthetic CTB gene into the chromosomal DNA was confirmed by PCR amplification analysis. A high level of CTB (2.1% of total soluble protein) was expressed in the endosperm tissue of the transgenic rice plants. The synthetic CTB produced only in the rice endosperm demonstrated strong affinity for GM1-ganglioside, thereby suggesting that the CTB subunits formed an active pentamer. The successful expression of CTB genes in transgenic plants makes it a powerful tool for the development of a plant-derived edible vaccine.

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

Similar content being viewed by others

References

  1. Daniell, H., Streatfield, S. J., & Wycoff, K. (2001). Medical molecular farming: Production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends in Plant Science, 6, 219–226. doi:10.1016/S1360-1385(01)01922-7.

    Article  CAS  Google Scholar 

  2. Mason, H. S., Warzecha, H., Mor, T., & Arntzen, C. J. (2002). Edible plant vaccines: Applications for prophylactic and therapeutic molecular medicine. Trends in Molecular Medicine, 8, 324–329. doi:10.1016/S1471-4914(02)02360-2.

    Article  CAS  Google Scholar 

  3. Walmsley, A. M., & Arntzen, C. J. (2003). Plant cell factories and mucosal vaccines. Current Opinion in Biotechnology, 14, 145–150. doi:10.1016/S0958-1669(03)00026-0.

    Article  CAS  Google Scholar 

  4. Faruque, S. M., Biswas, K., Udden, S. M., Ahmad, Q. S., Sack, D. A., Nair, G. B., et al. (2006). Transmissibility of cholera: In vivo-formed biofilms and their relationship to infectivity and persistence in the environment. Proceedings of the National Academy of Sciences of the United States of America, 103, 6350–6355. doi:10.1073/pnas.0601277103.

    Article  CAS  Google Scholar 

  5. Kaper, J. B., Morris, G., & Levine, M. M. (1995). Cholera. Clinical Microbiology Reviews, 8, 316.

    Google Scholar 

  6. Giddings, G. (2001). Transgenic plants as protein factories. Current Opinion in Biotechnology, 12, 450–454. doi:10.1016/S0958-1669(00)00244-5.

    Article  CAS  Google Scholar 

  7. Sixma, T. K., Pronk, S. E., Kalk, K. H., Wartna, E. S., van Zanten, B. A., Witholt, B., et al. (1991). Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli. Nature, 351, 371–377. doi:10.1038/351371a0.

    Article  CAS  Google Scholar 

  8. Zhang, R. G., Scott, D. L., Westbrook, M. L., Nance, S., Spangler, B. D., Shipley, G. G., et al. (1995). The three-dimensional crystal-structure of cholera toxin. Journal of Molecular Biology, 251, 563–573. doi:10.1006/jmbi.1995.0456.

    Article  CAS  Google Scholar 

  9. Holmgren, J., Adamsson, J., Anjuère, J., Clemens, J., Czerkinsky, C., Eriksson, K., et al. (2005). Mucosal adjuvants and anti-infection and anti-immunopathology vaccines based on cholera toxin, cholera toxin B subunit and CpG DNA. Immunology Letters, 97, 181–188. doi:10.1016/j.imlet.2004.11.009.

    Article  CAS  Google Scholar 

  10. Field, M., Rao, M. C., & Chang, E. B. (1989). Intestinal electrolyte transport and diarrheal disease. The New England Journal of Medicine, 321, 800–806.

    CAS  Google Scholar 

  11. Mckenzie, S. J., & Halsey, J. F. (1984). Cholera toxin-B subunit as a carrier protein to stimulate a mucosal immune-response. Journal of Immunology (Baltimore, Md.: 1950), 133, 1818–1824.

    CAS  Google Scholar 

  12. Czerkinsky, C., Russell, M. W., Lycke, N., Linblad, M., & Holmgren, J. (1989). Oral-administration of a streptococcal antigen couples to cholera-toxin B-subunit evokes strong antibody-responses in salivary-glands and extramucosal tissues. Infection and Immunity, 57, 1072–1077.

    CAS  Google Scholar 

  13. Dertzbaugh, M. T., & Elson, C. O. (1993). Reduction in oral immunogenicity of cholera toxin-B subunit by N-terminal peptide addition. Infection and Immunity, 61, 384–390.

    CAS  Google Scholar 

  14. Elson, C. O. (1989). Cholera toxin and its subunits as potential oral adjutants. Current Topics in Microbiology and Immunology, 146, 29–33.

    CAS  Google Scholar 

  15. Merritt, E. A., Sarfaty, S., Feil, I. K., & Hol, W. G. J. (1997). Structural foundation for the design of receptor antagonists targeting Escherichia coli heat-labile enterotoxin. Structure (London, England), 5, 1485–1499. doi:10.1016/S0969-2126(97)00298-0.

    CAS  Google Scholar 

  16. Butow, B. J., Ma, W., Gale, K. R., Cornish, G. B., Rampling, L., Larroque, O., et al. (2003). Molecular discrimination of Bx7 alleles demonstrates that a highly expressed high-molecular-weight glutenin allele has a major impact on wheat flour dough strength. Theoretical and Applied Genetics, 107, 1524–1532. doi:10.1007/s00122-003-1396-8.

    Article  CAS  Google Scholar 

  17. Butow, B. J., Gale, K. R., Ikea, J., Juhasz, A., Bedo, Z., Tamas, L., et al. (2004). Dissemination of the highly expressed Bx7 glutenin subunit (Glu-B1al allele) in wheat as revealed by novel PCR markers and RP-HPLC. Theoretical and Applied Genetics, 109, 1525–1535. doi:10.1007/s00122-004-1776-8.

    Article  CAS  Google Scholar 

  18. Shewry, P. R., Tatham, A. S., & Halford, N. G. (1999). The prolamins of the Triticeae. In R. Casey & P. R. Shewry (Eds.), Seed proteins (pp. 35–78). Kluwer Academic Press: Doldrecht.

    Google Scholar 

  19. Lamacchia, C., Shewry, P. R., Di Fonzo, N., Forsyth, J. L., Harris, N., Lazzeri, P. A., et al. (2001). Endosperm-specific activity of a storage protein gene promoter in transgenic wheat seed. Journal of Experimental Botany, 52, 234–250. doi:10.1093/jexbot/52.355.243.

    Article  Google Scholar 

  20. Oszvald, M., Kang, T. J., Jenes, B., Kim, T. G., Tamas, L., & Yang, M. S. (2007). Synthesis and assembly of Escherichia coli heat-labile enterotoxin B subunit in transgenic rice (Oryza sativa L.). Biotechnology and Bioprocessing Engineering, 12, 676–686.

    Article  CAS  Google Scholar 

  21. McElroy, D., Blowers, A. D., Jenes, B., & Wu, R. (1991). Construction of expression vectors based on the rice actin 1 (Act1) 5’ region for use in monocot transformation. Molecular and General Genetics, 231, 150–160. doi:10.1007/BF00293832.

    Article  CAS  Google Scholar 

  22. Li, Y. Z., Ma, H. M., Zhang, J. Z., Wang, Z. Y., & Hong, M. M. (1995). Effects of the first intron of rice waxy gene on the expression of foreign genes in rice and tobacco protoplasts. Plant Science, 108, 181–190. doi:10.1016/0168-9452(95)04143-I.

    Article  CAS  Google Scholar 

  23. Oszvald, M., Gardonyi, M., Tamas, C., Takacs, I., Jenes, B., & Tamas, L. (2008). Development and characterization of a chimaeric tissue specific promoter in wheat and rice endosperm. In Vitro Cellular & Developmental Biology. Plant, 44, 1–7. doi:10.1007/s11627-007-9082-1.

    Article  CAS  Google Scholar 

  24. Wang, X. G., Zhang, G. H., Liu, C. X., Zhang, Y. H., Xiao, C. Z., & Fang, R. X. (2001). Purified cholera toxin B subunit from transgenic tobacco plants possesses authentic antigenicity. Biotechnology and Bioengineering, 72, 490–494. doi:10.1002/1097-0290(20010220)72:4 ≤ 490::AID-BIT1011 ≥ 3.0.CO;2-0.

    Article  CAS  Google Scholar 

  25. Li, D., O’Leary, J., Huang, Y., Huner, N. P. A., Jevnikar, A. M., & Ma, S. W. (2006). Expression of cholera toxin B subunit and the B chain of human insulin as a fusion protein in transgenic tobacco plants. Plant Cell Reports, 25, 417–424. doi:10.1007/s00299-005-0069-2.

    Article  CAS  Google Scholar 

  26. Daniell, H., Lee, S. B., Panchal, T., & Wiebe, P. O. (2001). Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. Journal of Molecular Biology, 311, 1001–1009. doi:10.1006/jmbi.2001.4921.

    Article  CAS  Google Scholar 

  27. Molina, A., Hervas-Stubbs, S., Daniell, H., Mingo-Castel, A. M., & Veramendi, J. (2004). High-yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts. Plant Biotechnology Journal, 2, 141–153. doi:10.1046/j.1467-7652.2004.00057.x.

    Article  CAS  Google Scholar 

  28. Arakawa, T., Chong, D. K., Merritt, J. L., & Langridge, W. H. (1997). Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Research, 6, 403–413. doi:10.1023/A:1018487401810.

    Article  CAS  Google Scholar 

  29. Kim, T. G., Galloway, D. R., & Langridge, W. H. R. (2004). Synthesis and assembly of anthrax lethal factor-cholera toxin B-subunit fusion protein in transgenic potato. Molecular Biotechnology, 28, 175–183. doi:10.1385/MB:28:3:175.

    Article  CAS  Google Scholar 

  30. Choi, N. W., Estes, M. K., & Langridge, W. H. R. (2005). Synthesis and assembly of a cholera toxin B subunit-rotavirus VP7 fusion protein in transgenic potato. Molecular Biotechnology, 31, 193–2002. doi:10.1385/MB:31:3:193.

    Article  CAS  Google Scholar 

  31. Jiang, X. L., He, Z. M., Peng, Z. Q., Qi, Y., Chen, Q., & Yu, S. Y. (2007). Cholera toxin B protein in transgenic tomato fruit induces systemic immune response in mice. Transgenic Research, 16, 169–175. doi:10.1007/s11248-006-9023-5.

    Article  CAS  Google Scholar 

  32. Sharma, M. K., Singh, N. K., Jani, D., Sisodia, R., Thungapathra, M., Gautam, J. K., et al. (2008). Expression of toxin co-regulated piles subunit A (TCPA) of Vibrio cholera and its immunogenic epitopes fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum). Plant Cell Reports, 27, 307–318. doi:10.1007/s00299-007-0464-y.

    Article  CAS  Google Scholar 

  33. Rosales-Mendoza, S., Soria-Guerra, R. E., Olivera-Flores, M. T. D., Lopez-Revilla, R., Argullo-Astorga, G. R., & Jimenez-Bremont, J. F. (2007). Expression of Escherichia coli heat-labile enterotoxin b subunit (LTB) in carrot (Daucus carota L.). Plant Cell Reports, 26, 969–976. doi:10.1007/s00299-007-0310-2.

    Article  CAS  Google Scholar 

  34. Kim, T. G., Kim, M. Y., Kim, B. G., Kang, T. J., Kim, Y. S., Jang, Y. S., et al. (2007). Synthesis and assembly of Escherichia coli heat-labile enterotoxin B subunit in transgenic lettuce (Lactuca sativa). Protein Expression and Purification, 51, 22–27. doi:10.1016/j.pep. 2006.05.024.

    Article  CAS  Google Scholar 

  35. Athwal, D. S. (1971). Semidwarf rice and wheat in global food needs. The Quarterly Review of Biology, 4, 1–34. doi:10.1086/406754.

    Article  Google Scholar 

  36. Pelham, H. R. B. (1988). Evidence that luminal ER proteins are sorted from secreted proteins in a post-ER compartment. The EMBO Journal, 7, 913–918.

    CAS  Google Scholar 

  37. Kozak, M. (1989). The scanning model for translation: An update. The Journal of Cell Biology, 108, 229–241. doi:10.1083/jcb.108.2.229.

    Article  CAS  Google Scholar 

  38. Kang, T. J., Loc, N. H., Jang, M. O., & Yang, M. S. (2004). Modification of the cholera toxin B subunit coding sequence to enhance expression in plants. Molecular Breeding, 13, 143–153. doi:10.1023/B:MOLB.0000018762.27841.7a.

    Article  CAS  Google Scholar 

  39. Kang, T. J., Han, S. C., Jang, M. O., Kang, K. H., Jang, Y. S., & Yang, M. S. (2004). Enhanced expression of B-subunit of Escherichia coli heat-labile enterotoxin in tobacco by optimization of coding sequence. Applied Biochemistry and Biotechnology, 117, 175–187. doi:10.1385/ABAB:117:3:175.

    Article  CAS  Google Scholar 

  40. Nagy, Z. B., Varga-Orvos, Z., Szakal, B., Tamas, L., & Puskas, L. G. (2006). Assembling and cloning genes for fusion proteins using reverse transcription one-step overlap extension PCR method. Analytical Biochemistry, 351, 311–313. doi:10.1016/j.ab.2006.01.049.

    Article  CAS  Google Scholar 

  41. Chu, C. C., Wang, C. C., Sun, C. S., Hsu, C., Yin, K. C., & Chu, C. Y. (1975). Established an efficient medium for another culture of rice though competitive experiments on the nitrogen sources. Scientia Sinica, 18, 659–668.

    Google Scholar 

  42. Christou, P. (1997). Rice transformation: Bombardment. Plant Molecular Biology, 35, 197–203. doi:10.1023/A:1005791230345.

    Article  CAS  Google Scholar 

  43. Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Plant Physiology, 15, 473–497. doi:10.1111/j.1399-3054.1962.tb08052.x.

    Article  CAS  Google Scholar 

  44. Kang, T. J., & Yang, M. S. (2004). Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants. BMC Biotechnology, 4, 20. doi:10.1186/1472-6750-4-20.

    Article  Google Scholar 

  45. Li, Z., & Trick, H. N. (2005). Rapid method for high-quality RNA isolation from seed endosperm containing high levels of starch. BioTechniques, 38, 872–876.

    Article  CAS  Google Scholar 

  46. Dean, C., Jones, J., Favreau, M., Dunsmuir, P., & Bedbrook, J. (1988). Influence of flanking sequences on variability in expression levels of an introduced gene in transgenic tobacco plants. Nucleic Acids Research, 16, 9267–9283. doi:10.1093/nar/16.19.9267.

    Article  CAS  Google Scholar 

  47. Peach, C., & Velten, J. (1991). Transgene expression variability position effect of CAT and GUS reporter genes driven by linked divergent T-DNA promoters. Plant Molecular Biology, 17, 49–60. doi:10.1007/BF00036805.

    Article  CAS  Google Scholar 

  48. Schon, A., & Freire, E. (1989). Thermodynamics of intersubunit interactions in cholera toxin upon binding to the oligosaccharide portion of its cell surface receptor, ganglioside GM1. Biochemistry, 28, 5019–5024. doi:10.1021/bi00438a017.

    Article  CAS  Google Scholar 

  49. Oszvald, M., Kang, T. J., Tomoskozi, S., Tamas, C., Tamas, L., Kim, T. G., et al. (2007). Expression of a synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat labile enterotoxin in rice endosperm. Molecular Biotechnology, 35, 215–223. doi:10.1007/BF02686007.

    Article  CAS  Google Scholar 

  50. Jani, D., Meena, L. S., Rizwan-ul-Haq, Q. M., Singh, Y., Sharma, A. K., & Tyagi, A. K. (2002). Expression of cholera toxin B subunit in transgenic tomato plants. Transgenic Research, 11, 447–454. doi:10.1023/A:1020336332392.

    Article  CAS  Google Scholar 

  51. Wiley, P. R., Tosi, P., Evrard, A., Lovegrove, A., Jones, H. D., & Shewry, P. R. (2007). Promoter analysis and immunolocalisation show that puroindoline genes are exclusively expressed in starchy endosperm cells of wheat grain. Plant Molecular Biology, 64, 125–136. doi:10.1007/s11103-007-9139-x.

    Article  CAS  Google Scholar 

  52. Yamagata, H., Tamura, K., Tanaka, K., & Kasai, Z. (1986). Cell-free synthesis of rice prolamin. Plant and Cell Physiology, 27, 1419–1422.

    CAS  Google Scholar 

  53. Hood, E. E., Witcher, D. R., & Maddoock, S. (1997). Commercial production of avidin from transgenic maize: Characterization of transformant, production, processing, extraction and purification. Molecular Breeding, 3, 291–306. doi:10.1023/A:1009676322162.

    Article  CAS  Google Scholar 

  54. Doran, P. M. (2006). Foreign protein degradation and instability in plants and plant tissue cultures. Trends in Biotechnology, 24, 426–432.

    Article  CAS  Google Scholar 

  55. Fischer, R., & Emans, N. (2000). Molecular farming of pharmaceutical proteins. Transgenic Research, 9, 279–299. doi:10.1023/A:1008975123362.

    Article  CAS  Google Scholar 

  56. Bowman, C. C., & Clements, J. D. (2001). Differential biological and adjuvant activities of cholera toxin and Escherichia coli heat-labile enterotoxin hybrids. Infection and Immunity, 69, 1528–1535. doi:10.1128/IAI.69.3.1528-1535.2001.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This research was supported by the International Cooperation Program of the Ministry of Science and Technology, Republic of Korea, and the Bilateral Intergovernmental Science & Technology Cooperation, KOR 13/99. This study was also supported by the Hungarian Scientific Research Fund (OTKA T 034791) and, in part, by ICGEB (CRP/HUN00-02).

Author information

Authors and Affiliations

  1. Department of Biochemistry and Food Technology, Budapest University of Technology and Economics, Budapest, Hungary

    Maria Oszvald & Sandor Tomoskozi

  2. Department of Plant Physiology and Molecular Plant Biology, Eotvos Lorand University, Budapest, Hungary

    Maria Oszvald

  3. Department of Food and Nutrition, Chonbuk National University, Jeonju, 561-756, South Korea

    Tae-Jin Kang & Youn-Soo Cha

  4. Agricultural Biotechnology Center, Godollo, Hungary

    Barnabas Jenes & Laszlo Tamas

  5. Division of Biological Science and the Research Center for Bioactive Materials, Chonbuk National University, 664-14, Deokjin-Dong, Deokjin-Ku, Jeonju, 561-756, South Korea

    Tae-Geum Kim & Moon-Sik Yang

Authors
  1. Maria Oszvald
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tae-Jin Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sandor Tomoskozi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Barnabas Jenes
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tae-Geum Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Youn-Soo Cha
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Laszlo Tamas
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Moon-Sik Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Moon-Sik Yang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Oszvald, M., Kang, TJ., Tomoskozi, S. et al. Expression of Cholera Toxin B Subunit in Transgenic Rice Endosperm. Mol Biotechnol 40, 261–268 (2008). https://doi.org/10.1007/s12033-008-9083-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-008-9083-2

Keywords

Search

Navigation