Abstract
The cholera toxin B subunit (CTB) contains five identical polypeptides and targets glycosphingolipid receptors on eukaryotic cell surfaces. Increased expression of CTB in plants is critical for the development of edible vaccines. In this study, the coding sequence of the CTB gene was optimized, based on the modification of codon usage to that of tobacco plant genes and the removal of mRNA-destabilizing sequences. The synthetic CTB gene was cloned into a plant expression vector and expressed in tobacco plants under the control of the CaMV 35S promoter. The recombinant CTB protein constituted approximately 1.5% of the total soluble protein in transgenic tobacco leaves. This level of CTB production was approximately 15-fold higher than that in tobacco plants that were transformed with the bacterial CTB gene. The recombinant CTB produced by tobacco plants demonstrated strong affinity for GM1-ganglioside, which indicates that the sites required for binding and proper folding of the pentameric CTB structure were conserved. This is the first report on the optimization of the CTB-coding sequence to give a dramatic increase in CTB expression in plants.
Article PDF
Similar content being viewed by others
References
Adang M.J., Brody M.S., Cardineau G., Eagan N., Roush R.T., Shewmaker C.K., Jones A., Oakes J.V. and McBride K.E. 1993. The reconstruction and expression of a Bacillus thuringiensis cryIIIA gene in protoplasts and potato plants. Plant Mol. Biol. 21: 1131–1145.
Arakawa T., Chong D.K., Merritt J.L. and Langridge W.H. 1997. Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Res. 6: 403–413.
Arakawa T., Chong D.K. and Langridge W.H. 1998a. Efficacy of a food plant-based oral cholera toxin B subunit vaccine. Nat. Biotechnol. 16: 292–297.
Arakawa T., Yu J., Chong D.K., Hough J., Engen P.C. and Langridge W.H. 1998b. A plant-based cholera toxin B subunit-insulin fusion protein protects against the development of autoimmune diabetes. Nat. Biotechnol. 16: 934–938.
Bergerot I., Ploix C., Petersen J., Moulin V., Rask C., Fabien N., Lindblad M., Mayer A., Czerkinsky C., Holmgren J. and Thivolet C. 1997. A cholera toxoid-insulin conjugate as an oral vaccine against spontaneous autoimmune diabetes. Proc. Natl. Acad. Sci. USA 94: 4610–4614.
Chen G.F. and Inouye M. 1990. Suppression of the negative effect of minor arginine codons on gene expression; preferential usage of minor codons within the first 25 codons of the Escherichia coli genes. Nucleic Acids Res. 18: 1465–1473.
Chen C-Y.A. and Shyu A-B. 1995. AU-rich elements: characterization and importance in mRNA degradation. Trends Biochem. Sci. 20: 465–470.
Daniell H., Lee S.B., Panchal T. and Wiebe P.O. 2001. Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J. Mol. Biol. 311: 1001–1009.
DeRocher E.J., Vargo-Gogola E.C., Diehn S.H. and Green P.J. 1998. Direct evidence for rapid degradation of Bacillus thuringiensis toxin mRNA as a cause of poor expression in plants. Plant Physiol. 117: 1445–1461.
Elson C.O. 1989. Cholera toxin and its subunits as potential oral adjuvants. Curr. Top. Microbiol. Immunol. 146: 29–33.
Farabaugh P.J. 1996. Programmed translational frameshifting. Microbiol. Rev. 60: 103–134.
Field M., Rao M.C. and Chang E.B. 1989. Intestinal electrolyte transport and diarrheal disease. N. Engl. J. Med. 321: 800–806.
Goldman E., Rosenberg A.H., Zubay G. and Studier F.W. 1995. Consecutive low-usage leucine codons block translation only when near the 5' end of a message in Escherichia coli. J. Mol. Biol. 245: 467–473.
Haffani Y.Z., Overney S., Yelle S., Bellemare G. and Belzile F.J. 2000. Premature polyadenylation contributes to the poor expression of the Bacillus thuringiensis cry3Ca1 gene in transgenic potato plants. Mol. Gen. Genet. 264: 82–88.
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.
Haseloff J., Siemering K.R., Prasher D.C. and Hodge S. 1997. Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark Arabidopsis plants brightly. Proc. Natl. Acad. Sci. USA 94: 2122–2127.
Holmgren J. and Svennerholm A.M. 1990. Development of oral vaccines against cholera and enterotoxinogenic Escherichia coli diarrhea. Scand. J. Infect. Dis. (Suppl.) 76: 47–53.
Horvath H., Huang J., Wong O., Kohl E., Okita T. and Kannangara C.G. 2000. The production of recombinant protein in transgenic barley grains. Proc. Natl. Acad. Sci. USA 97: 1914–1919.
Huang Z., Dry I., Webster D., Strugnell R. and Wesselingh S. 2001. Plant-derived measles virus hemagglutinin protein induces neutralizing antibodies in mice. Vaccine 19: 2163–2171.
Huang J., Sutliff T.D., Wu L., Nandi S., Benge K., Terashima M., Ralston A.H., Drohan W., Huang N. and Rodriguez R.L. 2001. Expression and purification of functional human alpha-1-antitrypsin from cultured plant cells. Biotechnol. Prog. 17: 126–133.
Huang J., Wu L., Yalda D., Adkins Y., Kelleher S.L., Crane M., Lonnerdal B., Rodriguez R.L. and Huang N. 2002. Expression of functional recombinant human lysozyme in transgenic rice cell culture. Transgenic Res. 11: 229–239.
Ikemura T. 1981. Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes. J. Mol. Biol. 146: 1–21.
Jani D., Meena L.S., Rizwan-ul-Haq Q.M., Singh Y., Sharma A.K. and Tyagi A.K. 2002. Expression of cholera toxin B subunit in transgenic tomato plants. Transgenic Res. 11: 447–454.
Jarvis P., Belzile F. and Dean C. 1997. Inefficient and incorrect processing of the Ac transposase transcript in iae1 and wild-type Arabidopsis thaliana. Plant J. 11: 921–931.
Kang T.J. and Fawley M.W. 1997. Variable (CA/GT)n simple sequence repeat DNA in the alga Chlamydomonas. Plant Mol. Biol. 35: 943–948.
Kang T.J., Loc N.H., Jang M.O., Jang Y.S. and Yang M.S. 2003. Expression of the B subunit of E. coli heat-labile enterotoxin in the chloroplasts of plants and its characterization. Transgenic. Res. (in press)
Kaper J.B., Morris J.G. and Levine M. 1995. Cholera. Clin. Microbiol. Rev. 8: 48–86.
Kozak M. 1989. The scanning model for translation: An update. J. Cell Biol. 108: 229–241.
Lee J.H., Kim N.S., Kwon T.H., Jang Y.S. and Yang M.S. 2001. Increased production of human granulocyte-macrophage colony stimulating factor (hGM-CSF) by the addition of stabilizing polymer in plant suspension cultures. J. Biotechnol. 96: 205–211.
Mason H.S., Haq T.A., Clements J.D. and Arntzen C.J. 1998. Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene. Vaccine 16: 1336–1343.
McGhee J.R., Mestecky J., Dertzbaugh M.T., Eldridge J.H., Hirasawa M., Kiyono H. 1992. The mucosal immune system: from fundamental concepts to vaccine development. Vaccine 10: 75–88.
Merritt E.A., Sarfaty S., van den Akker F., L'Hoir C., Martial J.A. and Hol W.G. 1994. Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide. Protein Sci. 3: 166–175.
Munro S. and Pelham H.R. 1987. A C-terminal signal prevents secretion of luminal ER proteins. Cell. 48: 899–907.
Murashige T. and Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473–497.
Murray E.E., Rocheleau T., Eberle M., Stock C., Sekar V. and Adang M. 1991. Analysis of unstable RNA transcripts of insecticidal crystal protein genes of Bacillus thuringiensis in transgenic plants and electroporated protoplasts. Plant Mol. Biol. 16: 1035–1050.
Nemchinov L.G., Liang T.J., Rifaat M.M., Mazyad H.M., Hadidi A. and Keith J.M. 2000. Development of a plant-derived subunit vaccine candidate against hepatitis C virus. Arch. Virol. 145: 2557–2573
Ohme-Takagi M., Taylor C.B., Newman T.C. and Green P.J. 1993. The effect of sequences with high AU content on mRNA stability in tobacco. Proc. Natl. Acad. Sci. USA 90: 11811–11815.
Pelham H.R. 1988. Evidence that luminal ER proteins are sorted from secreted proteins in a post-ER compartment. EMBO J. 7: 913–918.
Perlak F.J., Fuchs R.L., Dean D.A., McPherson S.L. and Fischhoff D.A. 1991. Modification of the coding sequence enhances plant expression of insect control protein genes. Proc. Natl. Acad. Sci. USA 88: 3324–3328.
Pizza M., Giuliani M.M., Fontana M.R., Monaci E., Douce G., Dougan G., Mills K.H., Rappuoli R. and Del Giudice G. 2001. Mucosal vaccines: non-toxic derivatives of LT and CT as mucosal adjuvants. Vaccine 19: 2534–2541.
Richter L.J., Thanavala Y., Arntzen C.J. and Mason H.S. 2000. Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nat. Biotechnol. 18: 1167–1171.
Rosenberg A.H., Goldman E., Dunn J.J., Studier F.W. and Zubay G. 1993. Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system. J. Bacteriol. 175: 716–722.
Rouwendal G.J., Mendes O., Wolbert E.J. and Douwe de Boer A. 1997. Enhanced expression in tobacco of the gene encoding green fluorescent protein by modification of its codon usage. Plant Mol. Biol. 33: 989–999.
Sadeghi H., Bregenholt S., Wegmann D., Petersen J.S., Holmgren J. and Lebens M. 2002. Genetic fusion of human insulin B-chain to the B-subunit of cholera toxin enhances in vitro antigen presentation and induction of bystander suppression in vivo. Immunology 106: 237–245.
Schon A. and 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.
Sixma T.K., Pronk S.E., Kalk K.H., Wartna E.S., van Zanten B.A., Witholt B. and Hol W.G. 1991. Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli. Nature 351: 371–377.
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 sub-unit. Proc. Natl. Acad. Sci. USA 93: 7196–7201.
Streatfield S.J., Jilka J.M., Hood E.E., Turner D.D., Bailey M.R., Mayor J.M., Woodard S.L., Beifuss K.K., Horn M.E., Delaney D.E., Tizard I.R. and Howard J.A. 2001. Plant-based vaccines: unique advantages. Vaccine 19: 2742–2748.
Tarkowski A., Sun J.B., Holmdahl R., Holmgren J. and Czerkinsky C. 1999. Treatment of experimental autoimmune arthritis by nasal administration of a type II collagen-cholera toxoid conjugate vaccine. Arthritis Rheum. 42: 1628–1634.
Thanaraj T.A. and Argos P. 1996. Ribosome-mediated translational pause and protein domain organization. Protein Sci. 5: 1594–1612.
Tuboly T., Yu W., Bailey A., Degrandis S., Du S., Erickson L. and Nagy E. 2000. Immunogenicity of porcine transmissible gastro-enteritis virus spike protein expressed in plants. Vaccine 18: 2023–2028.
Van Haute E., Joos H., Maes M., Warren G., Van Montagu M. and Schell J. 1983. Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO J. 2: 411–417.
Varenne S. and Lazdunski C. 1986. Effect of distribution of unfavourable codons on the maximum rate of gene expression by a heterologous organism. J. Theor. Biol. 120: 99–110.
Wandelt C.I., Khan M.R., Craig S., Schroeder H.E., Spencer D. and Higgins T.J. 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: 181–192.
Wang X.G., Zhang G.H., Liu C.X., Zhang Y.H., Xiao C.Z. and Fang R.X. 2001. Purified cholera toxin B subunit from transgenic tobacco plants possesses authentic antigenicity. Biotechnol. Bioeng. 72: 490–494.
Williams N.A., Hirst T.R. and Nashar T.O. 1999. Immune modulation by the cholera-like enterotoxins: from adjuvant to therapeutic. Immunol. Today 20: 95–101.
World Health Organization (WHO). 1998. Global programme for vaccines and immunization. WHO, Geneva, Switserland.
Yang S.-H, Moran D.L., Jia H.-W., Bicar E.H., Lee M. and Scott M.P. 2002. Expression of a synthetic porcine _-lactalbumin gene in the kernels of transgenic maize. Transgenic Res. 11: 11–20.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kang, TJ., Loc, NH., Jang, MO. et al. Modification of the cholera toxin B subunit coding sequence to enhance expression in plants. Molecular Breeding 13, 143–153 (2004). https://doi.org/10.1023/B:MOLB.0000018762.27841.7a
Issue Date:
DOI: https://doi.org/10.1023/B:MOLB.0000018762.27841.7a