Improving the baculovirus expression vector system with vankyrin‐enhanced technology

Abstract

The baculovirus expression vector system (BEVS) is a widely used platform for the production of recombinant eukaryotic proteins. However, the BEVS has limitations in comparison to other higher eukaryotic expression systems. First, the insect cell lines used in the BEVS cannot produce glycoproteins with complex-type N-glycosylation patterns. Second, protein production is limited as cells die and lyse in response to baculovirus infection. To delay cell death and lysis, we transformed several insect cell lines with an expression plasmid harboring a vankyrin gene (P-vank-1), which encodes an anti-apoptotic protein. Specifically, we transformed Sf9 cells, Trichoplusia ni High Five™ cells, and SfSWT-4 cells, which can produce glycoproteins with complex-type N-glycosylation patterns. The latter was included with the aim to increase production of glycoproteins with complex N-glycans, thereby overcoming the two aforementioned limitations of the BEVS. To further increase vankyrin expression levels and further delay cell death, we also modified baculovirus vectors with the P-vank-1 gene. We found that cell lysis was delayed and recombinant glycoprotein yield increased when SfSWT-4 cells were infected with a vankyrin-encoding baculovirus. A synergistic effect in elevated levels of recombinant protein production was observed when vankyrin-expressing cells were combined with a vankyrin-encoding baculovirus. These effects were observed with various model proteins including medically relevant therapeutic proteins. In summary, we found that cell lysis could be delayed and recombinant protein yields could be increased by using cell lines constitutively expressing vankyrin or vankyrin-encoding baculovirus vectors.

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

“…It was also reported that a vankyrin-enhanced technology improved the baculovirus expression vector system. This study found that cell lysis could be delayed and that recombinant protein yields could be increased by using cell lines constitutively expressing vankyrin or vankyrin-encoding baculovirus vectors (Steele et al, 2017). SmartBac, a new baculovirus system, was developed for large protein complex production (Zhai et al, 2019).…”

Advancements in molecular design and bioprocessing of recombinant adeno‐associated virus gene delivery vectors using the insect‐cell baculovirus expression platform

“…Expression of anti‐apoptotic proteins such as vankyrins has been shown to maintain more than 90% viability at 96 hpi in both Sf9 and Hi5 cells. [ 107 ] Similarly, the anti‐apoptotic p35 gene expression also showed a 4‐fold increase in GFP titer while maintaining the culture at more than 50% viability at approximately 120 hpi. [ 108 ] Incorporation of these advancements in the AAV production process and their effect on AAV yield could be of potential interest in the future.…”

Identifying parameters to improve the reproducibility of transient gene expression in High Five cells

“…For many years the baculovirus expression vector system (BEVS) has been successfully employed to produce large amounts of recombinant protein in insect cells [11–13]. The BEVS was further improved over time to more easily and faster generate recombinant baculovirus [14], by the elimination of viral protease genes [15], achieving higher viability during expression [16] and the option to insert multiple expression cassette into one vector-backbone [17]. In general, BEVS is considered safe as baculovirus cannot replicate in humans [18].…”