Stabilized baculovirus vector expressing a heterologous gene and GP64 from a single bicistronic transcript

Enhanced expression of recombinant proteins utilizing a modified baculovirus expression vector

“…Here the target gene was co-expressed with a baculoviral factor essential for infectivity, while its native gene was deleted from the baculovirus genome. Baculovirus expression vector with a bicistronic expression cassette expressing the gene of interest and an essential baculovirus native gene, GP64, was shown to lead to a stabilized and enhanced recombinant protein production [6]. However, this strategy is technically complicated.…”

“…As a result, with an increase in viral passage number, loss of bacmid transposed foreign genes located within these excision prone sites, has also been reported. On the basis of these observations, it has been hypothesized that the genomic deletions are more likely to occur in regions with a low ori density, a property attributed to the existing BAC vectors [6]. Keeping these observations in mind, we followed a simplified strategy of utilizing AcMNPV homologous region, having dual properties of an ori as well as an enhancer, to modify the existing bacmid-based BEVS.…”

“…As shown by Pijlman et al [5] insertion of an extra hr in the BAC vector leads to significant improvement in the genetic stability of adjacent sequences, resulting in prolonged and persistent protein expression. In addition, further maintenance of the BAC sequences appears to be dependent on the orientation of the inserted hr [5,6].…”

Gene gymnastics

“…Genes that are essential for propagation in cell culture and genes which are detrimental for foreign protein production were delineated by several research groups. [22][23][24][25][26][27][28] Based on our positive experiences with bottom-up design of plasmids and engineering of the baculovirus genome itself to improve protein production, we became interested in extending our reengineering concept to rewiring the entire baculovirus genome to maximize its performance. Redesigning and restructuring the baculovirus genome for enhanced high-throughput robotic routines, which is basically unfeasible with the BAC/Tn7 entry system.…”

Opportunities and challenges for the baculovirus expression system

“…When the expression of the heterologous gene (GFP) is directly coupled to the expression of the essential gene gp64 in a bicistronic expression cassette (F), where both genes are separated by an internal ribosome entry site (IRES), GFP levels remain higher upon continued viral passages (G); BV titers are given in TCID50 units/ml. (Data shown are from Pijlman et al (2001Pijlman et al ( , 2002Pijlman et al ( , 2004Pijlman et al ( , 2006; Copyright Elsevier and reused with permission of Elsevier; Fig 4E, which no selection pressure exists to be maintained and that does not contain an origin of DNA replication. Therefore, an extra copy of homologous region 1 (hr1) was inserted in this area (Fig.…”