title: Stabilized baculovirus vector expressing a heterologous gene and GP64 from a single bicistronic transcript.
abstract: The efficient scale-up of recombinant protein production in insect-cell bioreactors using baculovirus expression vectors is hampered by reductions in yield with increasing viral passage, the so-called passage effect. This phenomenon is characterized by the generation and subsequent accumulation of defective interfering baculoviruses (DIs), which interfere with the replication of genomically intact virus. A novel baculovirus expression vector is presented equipped with a bicistronic expression cassette that allows the simultaneous expression of the recombinant gene (GFP, first cistron) and an essential baculovirus gene (GP64, second cistron) from a single messenger RNA (mRNA). The translation of GP64 is mediated by an internal ribosome entry site (IRES) element from Rhopalosiphum padi virus (RhPV) while the native GP64 gene is deleted. In this way, a dominant selection pressure is placed on the entire bicistronic mRNA and hence on the maintenance of the foreign gene. The bicistronic expression vector was superior to the control baculovirus vector in that GFP expression remained at much higher levels upon continued virus passage. The versatility of this stabilized vector was demonstrated by its ability to propagate in a number of cell lines including Sf21, Sf9 and High Five cells. This novel baculovirus vector is especially valuable for large-scale recombinant protein production in insect-cell bioreactors where the number of viral passages is high.
authors.affiliation: Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands.
authors.forename: Gorben P
authors.initials: GP
authors.lastname: Pijlman
authors.forename: Els C
authors.initials: EC
authors.lastname: Roode
authors.forename: Xiaoxiang
authors.initials: X
authors.lastname: Fan
authors.forename: Lisa O
authors.initials: LO
authors.lastname: Roberts
authors.forename: Graham J
authors.initials: GJ
authors.lastname: Belsham
authors.forename: Just M
authors.initials: JM
authors.lastname: Vlak
authors.forename: Monique M
authors.initials: MM
authors.lastname: van Oers
mesh_terms: D016367:Baculoviridae / Q000502:physiology*; D005796:Genes / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D042822:Genomic Instability / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics; D014760:Viral Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D014779:Virus Replication / Q000502:physiology*
chemical_list: D014157:Transcription Factors; D014760:Viral Fusion Proteins