title: Expression of the dengue virus structural proteins in Pichia pastoris leads to the generation of virus-like particles.
abstract: We have expressed cDNA encoding the dengue virus structural proteins in Pichia pastoris by chromosomal integration of an expression cassette containing the dengue virus structural genes (CprME). The yeast recombinant E protein migrated during SDS-PAGE as a 65 kDa protein when analysed by Western blotting and radioimmunoprecipitation, which is the expected molecular mass for correctly processed and glycosylated E protein. Treatment with endoglycosidases showed that the recombinant E protein was modified by the addition of short mannose chains. The E protein migrated with a buoyant density of 1.13 g/cm3 when analysed using sucrose density gradient centrifugation. Spherical structures with an average diameter of 30 nm, whose morphology resembles dengue virions, were observed in the purified fractions using transmission electron microscopy. Furthermore, the virus-like particles were immunogenic in animals and were able to induce neutralizing antibodies. This is the first report that expression of the structural genes of a flavivirus in yeast is able to generate particulate structures that resemble virions.
authors.affiliation: Institute of Molecular and Cell Biology, National University of Singapore, Republic of Singapore. mcbsrj@leonis.nus.sg
authors.forename: R J
authors.initials: RJ
authors.lastname: Sugrue
authors.forename: J
authors.initials: J
authors.lastname: Fu
authors.forename: J
authors.initials: J
authors.lastname: Howe
authors.forename: Y C
authors.initials: YC
authors.lastname: Chan
mesh_terms: D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary; D003716:Dengue Virus / Q000302:isolation & purification / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D008358:Mannose / Q000032:analysis; D008970:Molecular Weight; D010843:Pichia / Q000821:virology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015678:Viral Structural Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D014771:Virion / Q000302:isolation & purification / Q000502:physiology*
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D015678:Viral Structural Proteins; C072281:E protein TH Sman, Dengue virus; D008358:Mannose