title: Molecular and process design for rotavirus-like particle production in Saccharomyces cerevisiae.
abstract: BACKGROUND
  Virus-like particles (VLP) have an increasing range of applications including vaccination, drug delivery, diagnostics, gene therapy and nanotechnology. These developments require large quantities of particles that need to be obtained in efficient and economic processes. Production of VLP in yeast is attractive, as it is a low-cost protein producer able to assemble viral structural proteins into VLP. However, to date only single-layered VLP with simple architecture have been produced in this system. In this work, the first steps required for the production of rotavirus-like particles (RLP) in S. cerevisiae were implemented and improved, in order to obtain the recombinant protein concentrations required for VLP assembly.
  
  
  RESULTS
  The genes of the rotavirus structural proteins VP2, VP6 and VP7 were cloned in four Saccharomyces cerevisiae strains using different plasmid and promoter combinations to express one or three proteins in the same cell. Performance of the best constructs was evaluated in batch and fed-batch cultures using a complete synthetic media supplemented with leucine, glutamate and succinate. The strain used had an important effect on recombinant protein concentration, while the type of plasmid, centromeric (YCp) or episomal (YEp), did not affect protein yields. Fed-batch culture of the PD.U-267 strain resulted in the highest concentration of rotavirus proteins. Volumetric and specific productivities increased 28.5- and 11-fold, respectively, in comparison with batch cultures. Expression of the three rotavirus proteins was confirmed by immunoblotting and RLP were detected using transmission electron microscopy.
  
  
  CONCLUSIONS
  We present for the first time the use of yeast as a platform to express multilayered rotavirus-like particles. The present study shows that the combined use of molecular and bioprocess tools allowed the production of triple-layered rotavirus RLP. Production of VLP with complex architecture in yeasts could lead to the development of new vaccine candidates with reduced restrictions by regulatory agencies, using the successful experience with other yeast-based VLP vaccines commercialized worldwide.
authors.affiliation: Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México.
authors.forename: William A
authors.initials: WA
authors.lastname: Rodríguez-Limas
authors.forename: Keith E J
authors.initials: KE
authors.lastname: Tyo
authors.forename: Jens
authors.initials: J
authors.lastname: Nielsen
authors.forename: Octavio T
authors.initials: OT
authors.lastname: Ramírez
authors.forename: Laura A
authors.initials: LA
authors.lastname: Palomares
mesh_terms: D002586:Cesium / Q000737:chemistry; D002712:Chlorides / Q000737:chemistry; D007700:Kinetics; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012401:Rotavirus / Q000235:genetics / Q000276:immunology; D022243:Rotavirus Vaccines / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D058425:Vaccines, Virus-Like Particle / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D002712:Chlorides; D022243:Rotavirus Vaccines; D058425:Vaccines, Virus-Like Particle; D002586:Cesium; C028019:cesium chloride