Secreted production of assembled Norovirus virus-like particles from Pichia pastoris

Abstract

BackgroundNorovirus virus-like particles (NoV VLPs) have recently been explored as potential vaccine platforms due to their ability to produce an effective immune response. Expression of the main structural protein, VP1, leads to formation of self-assembled particles with similar characteristics to the original virus. These NoV VLPs have been expressed in Escherichia coli, yeast and insect cells. Expression in E. coli and insect cells share downstream processing issues due to the presence of inclusion bodies or the need for numerous purification steps. NoV VLPs have also been produced in the yeast P. pastoris; however the protein was only expressed intracellularly.ResultsWe have successfully expressed and secreted the VP1 protein in the novel P. pastoris strain, Bg11, using the methanol inducible pJ912 expression vector, containing the cDNA of NoV VP1. Expression of the VP1 protein in Bg11 was carried out in a 1.5 L bioreactor resulting in a total yield of NoV VLPs greater than 0.6 g/L. NoV VLPs obtained from the culture supernatant were purified via ion-exchange chromatography, resulting in particles with a purity over 90%. The average size of the particles after purification was 40 nm. Transmission electron microscopy was used to visualize the morphology of the particles and saliva-binding assay confirmed that the NoV VLPs bind to Histo-Blood Group Antigens (HBGA).ConclusionsIn this study we describe the expression and characterization of fully assembled Norovirus virus-like particles obtained from P. pastoris. The particles are similar in size, morphology and binding capacity, as previously described, for the original NoV. Our results detail the successful expression and secretion of VLPs in P. pastoris, improving their candidacy as a vaccine platform.

Rapid and sensitive detection of norovirus antibodies in human serum with a biolayer interferometry biosensor

“…Because the biological characterisation of human NoV has been hampered by the lack of an appropriate cell culture system and animal model for the propagation of the virus, virus-like particles (VLPs) have been used extensively to study NoV structure and stability, host-cell interactions, and as a tool in diagnostic serological assays [10][11][12][13]. Structural studies have shown that the noroviral capsid is composed almost entirely of the 58 kDa VP1 capsid protein [13], which self-assembles into empty capsids called virus-like particles (VLPs) when expressed recombinantly in insect cells or in yeast [14]. In this study, we utilised recently developed His-tagged VLPs (Koho et al, [31]), which are based on norovirus VLPs described earlier [15].…”

His-tagged norovirus-like particles: A versatile platform for cellular delivery and surface display

“…secreted production of NoV VLPs from Pichia pastoris was described recently [20]. Also, NoV VLP-based vaccines are under active development.…”

Optimization of norovirus virus‐like particle production in Pichia pastoris using a real‐time near‐infrared bioprocess monitor

“…This work builds on our previous study demonstrating the expression and secretion of the norovirus capsid viral protein 1 (VP1) in P. pastoris resulting in self‐assembled non‐infectious NoV VLPs . Here, VP1 was genetically modified to display the NY‐ESO‐1 cancer testis antigen for recombinant expression in P. pastoris .…”