title: Secreted production of assembled Norovirus virus-like particles from Pichia pastoris.
abstract: BACKGROUND
  Norovirus 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.
  
  
  RESULTS
  We 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).
  
  
  CONCLUSIONS
  In 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.
authors.forename: Jaime
authors.initials: J
authors.lastname: Tomé-Amat
authors.forename: Lauren
authors.initials: L
authors.lastname: Fleischer
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Parker
authors.forename: Cameron L
authors.initials: CL
authors.lastname: Bardliving
authors.forename: Carl A
authors.initials: CA
authors.lastname: Batt
mesh_terms: D001789:Blood Group Antigens / Q000378:metabolism; D006801:Humans; D008027:Light; D029322:Norovirus / Q000378:metabolism* / Q000648:ultrastructure; D010316:Particle Size; D010843:Pichia / Q000302:isolation & purification / Q000378:metabolism*; D012463:Saliva; D012542:Scattering, Radiation; D014771:Virion / Q000378:metabolism* / Q000648:ultrastructure; D019065:Virus Assembly
chemical_list: D001789:Blood Group Antigens