Abstract
A gene encoding the cholera toxin B subunit protein (CTB), fused to an endoplasmic reticulum (ER) retention signal (SEKDEL) was inserted adjacent to the bi-directional mannopine synthase P2 promoter in a plant expression vector containing a bacterial luciferase AB fusion gene (luxF) linked to the P1 promoter. Potato leaf explants were transformed by Agrobacterium tumefaciens carrying the vector and kanamycin-resistant plants were regenerated. The CTB-SEKDEL fusion gene was identified in the genomic DNA of bioluminescent plants by polymerase chain reaction amplification. Immunoblot analysis indicated that plant-derived CTB protein was antigenically indistinguishable from bacterial CTB protein, and that oligomeric CTB molecules (Mr ∼ 50 kDa) were the dominant molecular species isolated from transgenic potato leaf and tuber tissues. Similar to bacterial CTB, plant-synthesized CTB dissociated into monomers (Mr ∼ 15 kDa) during heat or acid treatment. The maximum amount of CTB protein detected in auxin-induced transgenic potato leaf and tuber tissues was approximately 0.3% of total soluble plant protein. Enzyme-linked immunosorbent assay methods indicated that plant-synthesized CTB protein bound specifically to GM1-ganglioside, the natural membrane receptor of cholera toxin. In the presence of the SEKDEL signal, CTB protein accumulates in potato tissues and is assembled into an oligomeric form that retains native biochemical and immunological properties. The expression of oligomeric CTB protein with immunological and biochemical properties identical to native CTB protein in edible plants opens the way for preparation of inexpensive food plant-based oral vaccines for protection against cholera and other pathogens in endemic areas throughout the world
Similar content being viewed by others
References
Block, M. de (1988) Genotype-independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens. Theor. Appl. Genet. 76, 767–74.
Chong, D.K.X., Roberts, W., Arakawa, T., Illes, K., Bagi, G., Slattery, C.W. and Langridge, W.H.R. (1997) Expression of the human milk protein, â-casein in transgenic potato plants. Transgenic Res. 6, 289–96.
Czerkinsky, C., Russell, M.W., Lycke, N., Lindbald, M. and Holmgren, J. (1989) Oral administration of a streptococcal antigen coupled to cholera toxin B subunit evokes strong antibody responses in salivary glands and extramucosal tissues. Infect. Immun 57, 1072–7.
Dertzbaugh, M.T. and Elson, C.O. (1993) Comparative effectiveness of the cholera toxin B subunit and alkaline phosphatase as carriers for oral vaccines. Infect. Immun. 61, 48–55.
Doyle, J.J. and Doyle, J.L. (1992) Isolation of plant DNA from fresh tissue. Focus 12, 13–15.
Elson, C.O. and Ealding, W. (1984) Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin. J. Immunol. 132, 2736–41.
Escher, A., O'Kane, D.J., Lee, J. and Szalay, A.A. (1989) Bacterial luciferase alpha-beta fusion protein is fully active as a monomer and highly sensitive in vivo to elevated temperature. Proc. Natl Acad. Sci. USA 86, 6528–32.
Fuhrman, J.A. and Cebra, J.J. (1981) Special features of the priming process for a secretory IgA response: B-cell priming with cholera toxin. J. Exp. Med. 153, 534–44.
Fujita, K. and Finkelstein, R.A. (1972) Antitoxic immunity in experimental cholera: comparison of immunity induced perorally and parenterally in mice. J. Infect. Dis. 125, 647–55.
Fukuta, S., Magnani, J.L., Twiddy, E.M., Holmes, R.K. and Ginsburg, V. (1988) Comparison of the carbohydrate-binding specificities of cholera toxin and Escherichia coli heat-labile enterotoxins LTh-1, LT-IIa, and LT-IIb. Infect. Immun. 56, 1748–53.
Gill, D.M. (1976) The arrangement of subunits in cholera toxin. Biochmistry 15, 1242–8.
Gill, D.M. and Meren, R. (1978) ADP-ribosylation of membrane proteins catalyzed by cholera toxin: basis of the activation of adenylate cyclase. Proc. Natl Acad. Sci. USA 75, 3050–4.
Hancock, W.W., Sayegh, M.H., Kwok, C.A., Weiner, H.L. and Carpenter, C.B. (1993) Oral, but not intravenous, alloantigen prevents accelerated allograft rejection by selective intragraft Th2 cell activation. Transplantation 55, 1112–18.
Haq, T.A., Mason, H.S., Clements, J.D. and Arntzen, C.J. (1995) Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science 268, 714–716.
Hardy, S.J.S., Holmgren, J., Johansson, S., Sanchez, J. and Hirst, T.R. (1988) Coordinated assembly of multisubunit proteins: oligomerization of bacterial enterotoxins in vivo and in vitro. Proc. Natl Acad. Sci. USA 85, 7109–13.
Hirst, T.R. and Holmgren, J. (1987) Conformation of protein secreted across bacterial outer membranes: a study of enterotoxin translocation from Vibrio cholerae. Proc. Natl Acad. Sci. USA 84, 7418–22.
Holmgren, J., Lycke, N. and Czerkinsky, C. (1993) Cholera toxin and cholera B subunit as oral mucosal adjuvant and antigen vector systems. Vaccine 11, 1179–84.
Jackson, R.J., Fujihashi, K., Xu-Amano, J., Kiyono, H., Elson, C.O. and McGhee, J.R. (1993) Optimizing oral vaccines: induction of systemic and mucosal B-cell and antibody responses to tetanus toxoid by use of cholera toxin as an adjuvant. Infect. Immun. 61, 4272–9.
Khoury, S.J., Lider, O., Al-Sabbagh, A. and Weiner, H.L. (1990) Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein. Cell. Immunol. 131, 302–10.
Koncz, C., Olsson, O., Langridge, W.H.R., Schell, J. and Szalay, A.A. (1987) Expression and assembly of functional bacterial luciferase in plants. Proc. Natl Acad. Sci. USA 84, 131–5.
Kozak, M. (1981) Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes. Nucleic Acid Res. 9, 5233–52.
Langridge, W.H.R., Fitzgerald, K.L., Koncz, C., Schell, J. and Szalay, A.A. (1989) Dual promoter of Agrobacterium tumefaciens mannopine synthase gene is regulated by plant growth hormones. Proc. Natl Acad. Sci. USA 86, 3219–23.
Langridge, W.H.R., Escher, A. nad Szalay, A.A. (1991) Measurement of bacterial luciferase as a reporter enzyme in vivo in transformed bacteria, yeast, plant cells and in transgenic plants. Technique 3, 99–108.
Lycke, N. and Holmgren, J. (1986) Strong adjuvant properties of cholera toxin on gut mucosal immune response to orally presented antigens. Immunology 59, 301–8.
Lycke, N., Lindholm, L. and Holmgren, J. (1983) IgA isotype restriction in the mucosal but not in the extramucosal immune response after oral immunizations with cholera toxin or cholera B subunit. Int. Arch. Allergy Appl. Immunol. 72, 119–27.
Lycke, N., Lindholm, L. and Holmgren, J. (1985) Cholera antibody production in vitro by peripheral blood lymphocytes following oral immunization of humans and mice. Clin. Exp. Immunol. 62, 39–47.
Ma, J.K.-C., Hiatt, A., Hein, M., Vine, N.D., Wang, F., Stabila, P., van Dolleweerd, D., Mostov, K. and Lehner, T. (1995) Generation and assembly of secretory antibodies in plants. Science 268, 716–19.
Mason, H.S. and Arntzen, C.J. (1995) Transgenic plants as vaccine production systems. Trends Biotechnol. 13, 388–92.
Mason, H.S., Lam, D.M.-K. and Arntzen, C.J. (1992) Expression of hepatitis B suface antigen in transgenic plants. Proc. Natl Acad. Sci. USA 89, 11745–9.
Mason, H.S., Ball, J.M., Shi, J.-J., Jiang, X., Estes, M.K. and Arntzen, C.J. (1996) Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc. Natl Acad. Sci. USA 93, 5335–40.
McKenzie, S.J. and Halsey, J.F. (1984) Cholera toxin B subunit as a carrier protein to stimulate a mucosal immune response. J. Immunol. 133, 1818–24.
Merritt, E.A., Sarfaty, S., Akker, F.V.-D., L'Hoir, C., Martial, J.A. and Hol, W.G.J. (1994) Crystal structure of cholera toxin Bpentamer bound to receptor GM1 pentasaccharide. Protein Sci. 3, 166–75.
Miller, A., Lider, O., Roberts, A.B., Sporn, M.B. and Weiner, H.L. (1992) Suppressor T cells generated by oral tolerization to myelin basic protein suppress both in vitro and in vivo immune responses by the release of transforming growth factor â after antigen-specific triggering. Proc. Natl Acad. Sci. USA 89, 421–5.
Munro, S. and Pelham, H.R.B. (1987) A C-terminal signal prevents secretion of luminal ER proteins. Cell 48, 899–907.
Sayegh, M.H., Khoury, S.J., Hancock, W.W., Weiner, H.L. and Carpenter, C.B. (1992) Induction of immunity and oral tolerance with polymorphic class II major histocompatibility complex allopeptides in the rat. Proc. Natl Acad. Sci. USA 89, 7762–6.
Schell, J. (1987) Transgenic plants as tools to study the molecular organization of plant genes. Science 237, 1176–83.
Schön, A. and Freire, E. (1989) Thermodynamics of intersubunit interactions in cholera toxin upon binding to the oligosaccharide portion of its cell suface receptor, ganglioside GM1, Biochemistry 28, 5019–24.
Sun, J.-B., Holmgren, J. and Czerkinsky, C. (1994) Cholera toxin B subunit: an efficient transmucosal carrier-delivery system for induction of peripheral immunological tolerance. Proc. Natl Acad. Sci. USA 91, 10795–9.
Sun, J.-B., Rask, C., Olsson, T., Holmgren, J. and Czerkinsky, C. (1996) Treatment of experimental autoimmune encephalomyelitis by feeding myelin basic protein conjugated to cholera toxin B subunit. Proc. Natl Acad. Sci. USA 93, 7196–201.
Svennerholm, L. (1976) Interaction of cholera toxin and ganglioside G(M1). Adv. Exp. Med. Biol. 71, 191–204.
Thanavala, Y., Yang, Y.-F., Lyons, P., Mason, H.S. and Arntzen, C.J. (1995) Immunogenicity of transgenic plant-derived hepatitis B surface antigen. Proc. Natl Acad. Sci. USA 92, 3358–61.
Trentham, D.E., Dynesius-Trentham, R.A., Orav, E.J., Combitchi, D., Lorenzo, C., Sewell, K.L., Hafler, D.A. and Weiner, H.L. (1993) Effects of oral administration of type II collagen on rheumatoid arthritis. Science 261, 1727–30.
Wandelt, C.I., Khan, M.R.I., Craig, S., Schroeder, H.E., Spencer, D. and Higgins, T.J.V. (1992) Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants. Plant J. 2, 181–92.
Weiner, H.L., Mackin, G.A., Matsui, M., Orav, E.J., Khoury, S.J., Dawson, D.M. and Hafler, D.A. (1993) Double-blind pilot trial or oral tolerization with myelin antigens in multiple sclerosis. Science 259, 1321–4.
Wolf, M.J.S. de, Fridkin, M., Epstein, M. and Kohn, L.D. (1981a) Structure-function studies of cholera toxin and its A and B protomers: modification of tryptophan residues. J. Biol. Chem. 256, 5481–8.
Wolf, M.J.S. de, Fridkin, M. and Kohn, L.D. (1981b) Tryptophan residues of cholera toxin and its A and B protomers: intrinsic fluorescence and solute quenching upon interacting with the ganglioside GM1, oligo-GM1, or dansylated oligo-GM1. J. Biol. Chem. 256, 5489–96.
Zhang, R.-G., Scott, D.L., Westbrook, M.L., Nance, S., Spangler, B.D., Shipley, G.G. and Westbrook, E.M. (1995) The threedimensional crystal structure of cholera toxin. J. Mol. Biol. 251, 563–73.
Zhang, Z.J., Davidson, L., Eisenbarth, G. and Weiner, H.L. (1991) Supression of diabetes in nonobese diabetic mice by oral administration of porcine insulin. Proc. Natl Acad. Sci. USA 88, 10252–6.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
ARAKAWA, T., CHONG, D.K., LAWRENCE MERRITT, J. et al. Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Res 6, 403–413 (1997). https://doi.org/10.1023/A:1018487401810
Issue Date:
DOI: https://doi.org/10.1023/A:1018487401810