Skip to main content

Advertisement

SpringerLink
Log in

Production of a fusion protein consisting of the enterotoxigenic Escherichia coli heat-labile toxin B subunit and a tuberculosis antigen in Arabidopsis thaliana

  • Genetic Transformation and Hybridization
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Transgenic plants are potentially safe and inexpensive vehicles to produce and mucosally deliver protective antigens. However, the application of this technology is limited by the poor response of the immune system to non-particulate, subunit vaccines. Co-delivery of therapeutic proteins with carrier proteins could increase the effectiveness of the antigen. This paper reports the ability of transgenic Arabidopsis thaliana plants to produce a fusion protein consisting of the B subunit of the Escherichia coli heat-labile enterotoxin and a 6 kDa tuberculosis antigen, the early secretory antigenic target ESAT-6. Both components of the fusion protein were detected using GM1-ganglioside-dependent enzyme-linked immunosorbant assay. This suggested the fusion protein retained both its native antigenicity and the ability to form pentamers.

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. 3a–c
Fig. 4
Fig. 5a, b
Fig. 6a, b
Fig. 7a, b
Fig. 8
Fig. 9

Similar content being viewed by others

Abbreviations

ELISA :

Enzyme linked immunosorbant assay

ESAT-6 :

Early secretory antigenic target (6 kDa)

ETEC :

Enterotoxigenic Escherichia coli

LTB :

B subunit of E. coli heat-labile enterotoxin

References

  • Bagdasarian MM, Nagai M, Frey J, Bagdasarian M (1999) Immunogenicity of Actinobacillus ApxIA toxin epitopes fused to the E. coli heat-labile enterotoxin B subunit. Vaccine 17:441–447

    Article  CAS  PubMed  Google Scholar 

  • Becker D, Kemper E, Schell J, Masterson R (1992) New plant binary vectors with selectable markers located proximal to the left T-DNA border. Plant Mol Biol 20:1195–1197

    CAS  PubMed  Google Scholar 

  • Brandt L, Oettinger T, Holm A, Andersen AB, Andersen P (1996) Key epitopes on the ESAT-6 antigen recognized in mice during the recall of protective immunity to Mycobacterium tuberculosis. J Immunol 157:3527–3533

    CAS  PubMed  Google Scholar 

  • Clements JD (1990) Construction of a nontoxic fusion peptide for immunization against Escherichia coli strains that produce heat-labile and heat-stable enterotoxins. Infect Immun 58:1159–1166

    CAS  PubMed  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  PubMed  Google Scholar 

  • Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300:1005–1006

    Article  CAS  PubMed  Google Scholar 

  • Haq TA, Mason HS, Clements JD, Arntzen CJ (1995) Oral immunization with a recombinant bacterial antigen produced in transgenic plant. Science 268:714–716

    Google Scholar 

  • Hwang SK, Kim YM (2000) A simple and reliable method for preparation of cross-contamination-free plant genomic DNA for PCR-based detection of transgenes. J Biochem Mol Biol 33:537–546

    CAS  Google Scholar 

  • Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti Plasmid. Nature 303:179–180

    CAS  Google Scholar 

  • Hol WGJ, Sixma TK, Merritt EA (1995) Structure and function of E. coli heat-labile enterotoxin and cholera toxin B pentamer. In: Moss J, Iglewski B, Vaughan M, Tu AT (eds) Bacterial toxins and virulence factors in disease. Dekker, New York, pp 185–223

  • Horwitz MA, Lee BE, Dillon BJ, Harth G (1995) Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis. Proc Natl Acad Sci USA 92:1530–1534

    CAS  PubMed  Google Scholar 

  • Horwitz MA, Dillon BJ, Galic SM (2000) Recombinant bacillus Calmette-Guerin (BCG) vaccines expressing the Mycobacterium tuberculosis 30-kDa major secretory protein induce greater protective immunity against tuberculosis than conventional BCG vaccines in a highly susceptible animal model. Proc Natl Acad Sci USA 97:13853–13858

    CAS  PubMed  Google Scholar 

  • Jagusztyn-Krynicka EK, Clark-Curtiss JE, Curtiss R 3rd (1993) Escherichia coli heat-labile toxin subunit B fusions with Streptococcus sobrinus antigens expressed by Salmonella typhimurium oral vaccine strains: importance of the linker for antigenicity and biological activities of the hybrid proteins. Infect Immun 61:1004–1015

    CAS  PubMed  Google Scholar 

  • Liljeqvist S, Stahl S, Andreoni C, Binz H, Uhlen M, Murby M (1997) Fusions to the cholera toxin B subunit: influence on pentamerization and GM1 binding. J Immunol Methods 210:125–135

    Article  CAS  PubMed  Google Scholar 

  • Lipscombe M, Charles IG, Roberts M, Dougan G, Tite J, Fairweather NF (1991) Intranasal immunization using the B subunit of the Escherichia coli heat-labile toxin fused to an epitope of the Bordetella pertussis P.69 antigen. Mol Microbiol 5:1385–1392

    CAS  PubMed  Google Scholar 

  • Mason HS, Haq TA, Clements JD, Arntzen CJ (1998) Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene. Vaccine 16:1336–1343

    Article  CAS  PubMed  Google Scholar 

  • Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6

    CAS  Google Scholar 

  • O’Dowd AM, Botting CH, Precious B, Shawcross R, Randall RE (1999) Novel modifications to the C-terminus of LTB that facilitate site-directed chemical coupling of antigens and the development of LTB as a carrier for mucosal vaccines. Vaccine 17:1442–1453

    Article  CAS  PubMed  Google Scholar 

  • Pollock JM, Andersen P (1997) Predominant recognition of the ESAT-6 protein in the first phase of interferon with Mycobacterium bovis in cattle. Infect Immun 65:2587–2592

    CAS  PubMed  Google Scholar 

  • Sandkvist M, Hirst TR, Bagdasarian M (1987) Alterations at the carboxyl terminus change assembly and secretion properties of the B subunit of Escherichia coli heat-labile enterotoxin. J Bacteriol 169:4570–4575

    CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp 7.43–7.48

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA Sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    PubMed  Google Scholar 

  • Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164:49–53

    Article  CAS  PubMed  Google Scholar 

  • Wada K, Aota S, Tsuchiya R, Ishibashi F, Gojobori T, Ikemura T (1990) Codon usage tabulated from the GenBank genetic sequence data. Nucleic Acids Res 18:2367–2411

    Google Scholar 

  • Walmsley AM, Alvarez ML, Jin Y, Kirk DD, Lee SM, Pinkhasov J, Rigano MM, et al (2003) Expression of the B subunit of Escherichia coli heat-labile enterotoxin as a fusion protein in transgenic tomato. Plant Cell Rep 21:1020–1026

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr. Hugh Mason of The Boyce Thompson Institute for supply of the basic vectors pLTB-L, TH110 and pGPTV.kan, Benchmark Biolabs Inc. for supply of the LTB standards and primary antibodies and the TB Research Materials and Vaccine Testing Contract at Colorado State University for supply of the ESAT-6 standards and primary antibodies. We thank Dr. Lokesh Joshi for his time and expertise in protein glycosylation. This work was supported by the internal competitive grant scheme of the Boyce Thompson Institute for Plant Research and Dow AgroSciences LLC. Dr. M. Lucrecia Alvarez is a Fellow from Fundación Antorchas (Argentina).

Author information

Authors and Affiliations

  1. School of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287–4501, USA

    M. L. Alvarez, J. Pinkhasov, Y. Jin, C. J. Arntzen & A. M. Walmsley

  2. Department of Biology, University of Milan, Milan, Italy

    M. M. Rigano & F. Sala

Authors
  1. M. M. Rigano
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. L. Alvarez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. Pinkhasov
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Y. Jin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. F. Sala
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. C. J. Arntzen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. A. M. Walmsley
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to A. M. Walmsley.

Additional information

Communicated by W.A. Parrott

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rigano, M.M., Alvarez, M.L., Pinkhasov, J. et al. Production of a fusion protein consisting of the enterotoxigenic Escherichia coli heat-labile toxin B subunit and a tuberculosis antigen in Arabidopsis thaliana . Plant Cell Rep 22, 502–508 (2004). https://doi.org/10.1007/s00299-003-0718-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-003-0718-2

Keywords

Search

Navigation