title: Metabolic engineering of the baculovirus-expression system via inverse "shotgun" genomic analysis and RNA interference (dsRNA) increases product yield and cell longevity.
abstract: RNA interference (RNAi) is as powerful tool for characterizing gene function in eukaryotic organisms and cultured cell lines. Its use in metabolic engineering has been limited and few reports have targeted protein expression systems to increase yield. In this work, we examine the use of in vitro synthesized double stranded RNA (dsRNA) in the baculovirus expression vector system (BEVS), using commercially relevant cultured cells (Spodoptera frugiperda, Sf-9) and larvae (Trichoplusia ni) as hosts. First, we employed an inverse "shotgun" genomic analysis to "find" an array of 16 putative insect gene targets. We then synthesized dsRNA in vitro targeting these genes and investigated the effects of injected dsRNA on larval growth, development, and product yield. Growth and development was at times stunted and in several cases, the effects were lethal. However, dsRNA targeting an acidic juvenile hormone-suppressible protein (AJHSP1), and translational elongation factor 2 (Ef-2) resulted in significantly increased yield of model product, GFP. Next, we targeted known genes, v-cath and apoptosis inducer, sf-caspase 1, in cultured Sf-9 cells. We confirm RNAi-mediated sf-caspase 1 suppression in Sf-9 cells, but not in baculovirus-infected cells, likely due to the overriding effects of inhibitor of apoptosis protein, p35. We also demonstrate suppression of v-cath in infected cells, which leads to a approximately 3-fold increase in product yield. Overall, our results support the application of RNAi in metabolic engineering, specifically for enhancing protein productivity in the baculovirus expression vector system.
authors.affiliation: Center for Biosystems Research, University of Maryland Biotechnology Institute, 5115 Plant Science Building, College Park, Maryland 20742, USA.
authors.forename: Eun Jeong
authors.initials: EJ
authors.lastname: Kim
authors.forename: Shannon F
authors.initials: SF
authors.lastname: Kramer
authors.forename: Colin G
authors.initials: CG
authors.lastname: Hebert
authors.forename: James J
authors.initials: JJ
authors.lastname: Valdes
authors.forename: William E
authors.initials: WE
authors.lastname: Bentley
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D049109:Cell Proliferation; D002470:Cell Survival; D016922:Cellular Senescence; D002874:Chromosome Mapping / Q000379:methods; D024861:Genetic Enhancement / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D034622:RNA Interference / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism*; D018411:Spodoptera / Q000166:cytology* / Q000502:physiology*
chemical_list: D011994:Recombinant Proteins