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Characterization of the Dengue Virus Envelope Glycoprotein Expressed in Pichia pastoris

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

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.

Key Words

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

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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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