Summary auto-generated
Researchers expressed dengue virus type 1 structural proteins (capsid, prM, and E) in the yeast Pichia pastoris using chromosomal integration of an expression cassette. The recombinant E protein was processed correctly and glycosylated with short mannose chains, migrating at the expected 65 kDa molecular weight. Sucrose density gradient centrifugation and transmission electron microscopy revealed the formation of spherical virus-like particles (VLPs) approximately 30 nm in diameter with morphology resembling dengue virions. These particles accumulated intracellularly with a buoyant density of 1.13 g/cm³. When purified VLPs were used to immunize rabbits, the resulting antiserum specifically recognized dengue virus E and prM proteins in infected cells and induced neutralizing antibodies against infectious dengue virus. This represents the first demonstration that expression of flavivirus structural genes in yeast generates particulate structures resembling virions. The Pichia pastoris system offers advantages over mammalian expression systems in scalability, productivity, and safety for potential pharmaceutical applications.
Key findings
- Expression of dengue virus structural genes (CprME) in Pichia pastoris produced virus-like particles approximately 30 nm in diameter with dengue virion-like morphology
- Recombinant E protein was correctly processed and modified by N-linked glycosylation with short mannose chains without hyperglycosylation
- Purified virus-like particles induced neutralizing antibodies against infectious dengue virus in immunized animals
- Yeast-derived virus-like particles accumulated intracellularly with a buoyant density of 1.13 g/cm³, similar to flaviviral VLPs from animal cell culture
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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.