Characterization of the Dengue Virus Envelope Glycoprotein Expressed in Pichia pastoris

  • Boon-Huan Tan
  • Jian Lin Fu
  • Richard J. Sugrue
Part of the Methods in Molecular Biology book series (MIMB, volume 379)


The full-length and truncated forms of recombinant envelope (E) glycoprotein from Dengue virus type 1, Singapore strain S275/90 were expressed in the yeast, Pichia pastoris, using a secretory vector. A truncated form of the E protein in which the transmembrane domain was deleted was secreted successfully into the culture medium. The E protein was also co-expressed with C and prM proteins using a non-secretory yeast vector. The co-expression of C, prM and E proteins resulted in the spontaneous formation of virus-like particles (VLPs), which were confirmed by sucrose gradient analysis and transmission electron microscopy. Furthermore, the VLPs were used to immunise rabbits, and shown to be immunogenic by immunofluorescence staining of dengue virus-infected Vero cells. The yeast-expressed E protein was treated with PNGase F, which showed that although the protein was modified by the addition of N-linked glycans, the recombinant expressed E protein was not hyperglycosylated.

Key Words

Dengue Pichia pastoris E glycoprotein CprME expression secretion glycosidase digestion virus-like-particles sucrose gradient 


  1. 1.
    Monath, T. P. (1994) Dengue: the risk to developed and developing countries. Proc. Natl. Acad. Sci. USA 91(7), 2395–2400.CrossRefPubMedGoogle Scholar
  2. 2.
    World Health Organisation (1997) Dengue Hemorrhagic Fever: Diagnosis, Treatment and Control, 2nd Ed., Geneva.Google Scholar
  3. 3.
    Tan, B-H., Yap, E-H., and Sugrue, R. J. (2004) Prospects for antiviral strategies in the treatment and prevention of Dengue virus infections. Curr. Topics Virol. 4, 19–37.Google Scholar
  4. 4.
    Fujita, H., Sumiyoshi, H., Mori, C., et al. (1987) Studies on the development of Japanese encephalitis vaccine: expression of virus envelope glycoprotein V3 (E) gene in yeast. Bulletin of the World Health Organization 65, 303–308.PubMedGoogle Scholar
  5. 5.
    Grinna, L. S. and Tscholl, J. F. (1989) Size, distribution and general structural features of N-linked oligosaccharides from the methylotropic yeast Pichia pastoris. Yeast 5, 107–115.CrossRefPubMedGoogle Scholar
  6. 6.
    Trimble, R. B., Atkinson, P. H., Tschopp, J. F., Townsend, R. R., and Maley, F. (1991) Structure of oligosaccharides on Saccharomyces SU2 invertase secreted by the methylotropic yeast Pichia pastoris. J. Biol. Chem. 266, 22,807–22,817.PubMedGoogle Scholar
  7. 7.
    Macauley-Patrick, S., Fazenda, M. L., McNeil, B., and Harvey, L. M. (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22(4), 249–270.CrossRefPubMedGoogle Scholar
  8. 8.
    Fu, J., Tan, B-H., Yap, E. H., Chan, Y. C., and Tan, Y. H. (1992) Full-length cDNA sequence of dengue type 1 virus (Singapore strain 275/90). Virology 188, 953–958.CrossRefPubMedGoogle Scholar
  9. 9.
    Sugrue, R. J., Cui, T., Xu, Q., Fu, J., and Chan, Y. C. (1997) The production of recombinant dengue virus E protein using Escherichia coli and Pichia pastoris. J. Virol. Meth. 69(1–2), 159–169.CrossRefGoogle Scholar
  10. 10.
    Sugrue, R. J., Fu, J., Howe, J., and Chan, Y. C. (1997) Expression of the dengue virus structural proteins in Pichia pastoris leads to the generation of virus-like particles. J. Gen. Viol. 78(8), 1861–1866.Google Scholar
  11. 11.
    Cregg, J. M., Vedvick, T. S., and Raschke, W. C. (1993) Recent advances in the expression of foreign genes in Pichia pastoris. BioTechnology II(8), 905–910.Google Scholar
  12. 12.
    Waterham, H. R., Digan, M. E., Koutz, P. J., Lair, S. V., and Cregg, J. M. (1997) Isolation of the Pichia. Gene 186(1), 37–44.CrossRefPubMedGoogle Scholar
  13. 13.
    Scorer, C. A., Buckholz, R. G., Clare, J. J., and Romanos, M. A. (1993) The intracellular production and secretion of HIV-1 envelope protein in the methylotropic ueast Pichia pastoris. Gene 136, 111–119.CrossRefPubMedGoogle Scholar
  14. 14.
    Konishi, E., Pincus, S., Paoletti, E., Shope, R. E., Burrage, T., and Mason, P. W. (1992) Mice immunised with a sub viral particle containing the Japanese encephalitis virus prM/M and E proteins are protected from lethal JEV infection. Virology 188, 714–720.CrossRefPubMedGoogle Scholar
  15. 15.
    Pincus, S., Mason, P. W., Konishi, E., et al. (199) Recombinant Vaccinia virus producing the prM and E proteins of yellow fever virus protects mice from lethal yellow fever encephalitis. Virology 187, 290–297.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Boon-Huan Tan
    • 1
  • Jian Lin Fu
    • 2
  • Richard J. Sugrue
    • 3
  1. 1.Virology Group Defence Medical and Environmental Research InstituteDSO National LaboratoriesSingapore
  2. 2.Transgenic Frog Facility, Institute of Molecular and Cell BiologyProteosSingapore
  3. 3.MRC Virology UnitInstitute of VirologyGlasgowUK

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