Abstract
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.
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Monath, T. P. (1994) Dengue: the risk to developed and developing countries. Proc. Natl. Acad. Sci. USA 91(7), 2395–2400.
World Health Organisation (1997) Dengue Hemorrhagic Fever: Diagnosis, Treatment and Control, 2nd Ed., Geneva.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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© 2007 Humana Press Inc., Totowa, NJ
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Tan, BH., Fu, J.L., Sugrue, R.J. (2007). Characterization of the Dengue Virus Envelope Glycoprotein Expressed in Pichia pastoris . In: Sugrue, R.J. (eds) Glycovirology Protocols. Methods in Molecular Biology, vol 379. Humana Press. https://doi.org/10.1007/978-1-59745-393-6_12
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DOI: https://doi.org/10.1007/978-1-59745-393-6_12
Publisher Name: Humana Press
Print ISBN: 978-1-58829-590-3
Online ISBN: 978-1-59745-393-6
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