Generation of baculovirus vectors for the high‐throughput production of proteins in insect cells

Abstract

The baculovirus expression system is one of the most popular methods used for the production of recombinant proteins but has several complex steps which have proved inherently difficult to adapt to a multi-parallel process. We have developed a bacmid vector that does not require any form of selection pressure to separate recombinant virus from non-recombinant parental virus. The method relies on homologous recombination in insect cells between a transfer vector containing a gene to be expressed and a replication-deficient bacmid. The target gene replaces a bacterial replicon at the polyhedrin loci, simultaneously restoring a virus gene essential for replication. Therefore, only recombinant virus can replicate facilitating the rapid production of multiple recombinant viruses on automated platforms in a one-step procedure. Using this vector allowed us to automate the generation of multiple recombinant viruses with a robotic liquid handler and then rapidly screen infected insect cell supernatant for the presence of secreted proteins.

MultiBac turns sweet

“…It is based on a modified AcNPV backbone that has a deletion in the essential gene orf1629 and contains a bacterial artificial chromosome (BAC) at the polyhedrin gene locus replacing polyhedrin gene. 20 The partial deletion of orf1629 renders the virus inactive and prevents replication of any non-recombinant parental virus in insect cells. Homologous recombination between a transfer and the flash-BAC backbone restores the function of the essential gene and allows for virus replication.…”

Genetic modification of a baculovirus vector for increased expression in insect cells

“…The baculovirus DNA AcRP30.AvrII 2 .bac (Possee et al 2008) was mixed with a transfer plasmid based on pBS.SK containing the lacZ coding region under the control of the polyhedrin gene promoter and flanked by approximately 1,000 bp from the AcMNPV genome (Ayres et al 1994) corresponding to nucleotides 104161-105207 and 107954-109080. These regions border the chiA and cath genes in AcMNPV.…”

“…Individual colonies were amplified in liquid medium containing 15 μg/ml chloramphenicol and 15 μg/ml kanomycin and bacmid DNA was isolated by alkaline lysis before being purified on caesium chloride gradients. The virus DNA was then further modified by digesting with AvrII and religating to remove part of ORF1629 (Possee et al 2008).…”

“…The virus DNAs used were BacPAK6 (contains chiA, cath, p26, p10 and p74) or bacmid-based derivatives of this virus, which have different gene deletions including Ac chiA− (deficient in chiA) and Ac chiA−/cath− (deficient in chiA and cath). Co-transfections were carried out in 1 × 10 6 Spodoptera frugiperda 9 (Sf9) cells in 35-mm 2 cell culture dishes as described by King and Possee (1992) or in 24-well plates (Possee et al 2008). Five days post-transfection, 0.5 ml cotransfection medium was added to 50 ml of Sf9 cells (2 × 10 6 cells/ml) and incubated for a further 5 days to produce a high-titre virus stock.…”

“…The system is based on the deletion of the virus polyhedrin gene (polh), which is replaced by the gene to be expressed, under the control of the powerful polh promoter. In recent years, the incorporation of bacterial replicons into the baculovirus genome has greatly improved this system, making it much simpler and faster to generate recombinant viruses (Luckow et al 1993;Possee et al 2008). The baculovirus most often used as an expression vector is Autographa californica multinucleopolyhedrovirus (AcMNPV).…”