Oxford Expression Technologies - flashBAC
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Information on the Oxford Expression Technologies flashBAC™ Baculovirus Protein Expression System
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flashBAC™ is a family products which use major new platform technology based on baculovirus protein expression technology. The technology enables fast and simultaneous production of multiple recombinant viruses, lending itself to use for high-throughput systems.
 
• SIMPLE - No plaque assays or tiresome selection procedures.
• STABLE - Genetically stable recombinant viruses for long-term expression
• SPEEDY - One-step, no tedious selection stages. Get you virus stock in 7-10 days
• SUPERIOR - Genetically optimised to delivery excellent protein yield and quality
Literature Description Size File
Quick Start Guide to the flashBAC System
 50KB Download flashBAC Quick Start Guide
flashBAC One-Step Protein Expression User Guiide 2.0MB Download Protocol
flashBAC Products
Go to Technical Overview - How flashBAC Works Technical Overview - How flashBAC Works
Go to flashBAC flashBAC
Go to flashBACGOLD flashBACGOLD
Go to flashBACULTRA flashBACULTRA
Specific Product/Application Areas:
Go to baculoQUANT Product Information baculoQUANT Virus Titration
Go to titrePLUS Product Information titrePLUS Virus Titration and Protein Expression
Go to pOET Transfer Plasmids Product Information pOET Transfer Vectors
Go to baculoFECTIN Transfection Reagent Information baculoFECTIN Transfection Reagent
Go to NEW Products NEW Products
Go to NEW superSf9 Cells NEW superSf9 Cells
Go to NEW Transfer Vectors Four NEW Transfer Vectors for C-Terminal and N-Terminal 6xHis-Tag® Fusion Sequences
Go to baculoFECTIN Transfection Reagent NEW baculoFECTIN Transfection Reagent
Go to NEW baculoGROW Serum-free Insect Medium NEW baculoGROW Serum-free Insect Medium
Go to NEW baculoCOMPLETE - ALL-IN-ONE Baculovirus Expression Kit NEW baculoCOMPLETE - ALL-IN-ONE Baculovirus Expression Kit
Go to NEW flashBAC™ Selection Box NEW flashBAC™ Selection Box
Go to NEW 96 Reaction flashBAC™ Kits NEW 96 Reaction flashBAC™ Kits
Literature
Go to flashBAC Technical Literature Download Oxford Expression Technologies Technical Literature
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Technical Overview - How flashBAC Works
The flashBAC system works by creating a recombinant baculovirus to over express the protein encoded by your ‘gene of interest’ in a cell line of your choice. The flashBAC, flashBACGOLD and flashBACULTRA products also contain gene deletions to help further enhance recombinant protein yield and quality.

A recombinant baculovirus is produced by simply co-transfecting insect cells with the flashBAC virus DNA supplied in the kit and a suitable transfer vector (such as a pOET transfer Vector) containing ‘the gene under investigation’. Homologous recombination within the insect cells (see diagram opposite) restores the function of an essential gene that is partly deleted in flashBAC (ORF1629), allowing the flashBAC virus DNA to replicate and produce virus particles. This also simultaneously inserts ‘the gene under investigation’ into the virus DNA under the control of the polyhedrin promoter. The recombinant virus genome, with the restored essential gene, replicates to produce baculovirus that can be harvested from the culture medium of the transfected insect cells (and forms a seed stock of recombinant virus). This recombinant virus is used to produce the protein.

As it is not possible for non-recombinant virus to replicate, there is no need for any selection system. Additionally a control vector is supplied with each kit so that you can double check that your co-transfection has worked. This one-step procedure greatly facilitates the high throughput production of baculovirus expression vectors via automated systems. However, it is also of benefit to the small research group just requiring one or a few recombinant baculoviruses prepared in individual dishes of cells.
To get started with the flashBAC system all that is needed is a flashBAC kit and a transfer vector (plasmid) into which the gene of interest has been cloned. Oxford Expression Technologies offer the range of pOET Transfer Vectors and the flashBAC system is also back compatible with all baculovirus transfer vectors based on homologous recombination in insect cells at the polyhedrin gene locus (see below for list of compatible transfer vectors).
Homologous recombination restores function of an essential gene allowing flashBAC virus DNA to replicate
Literature Description Size File
List of Compatible Transfer Vectors
 30KB Download List of Compatible Transfer Vectors
Product Technical Description
flashBAC™
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 The flashBAC™ system maximises protein secretion and membrane protein targeting. Baculovirus genomes contain several auxillary genes, which are non-essential for replication, including a chitinase (chiA), with exo- and endochitinase activity1. In an infected insect chitinase (together with cathepsin) facilitate host cuticle breakdown and tissue liquefaction at the very late stages of infection, so releasing the virus to infect more hosts1.

Confocal and electron microscopy observations of insect cells infected with AcMNPV have shown that the endoplasmic reticulum (ER) is densely packed with chitinase, completely blocking the secretory pathway. Deletion of chiA from flashBAC™ has improved the efficacy of the secretory pathway and resulted in a greatly enhanced (up to 60-fold in some instances) yield of recombinant proteins that are secreted or membrane targeted (in comparison with recombinant viruses that synthesise chitinase).

Note 1: Contact us for information on the references cited
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flashBACGOLD™
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 flashBACGOLD™ enhance yields for difficult to express proteins.

Baculovirus genomes contain several auxiliary genes, which are non-essential for replication in insect cell culture. Two of these are chitinase (chiA), which encodes an enzyme with exo- and endochitinase activity1 and a cathepsin-like cysteine protease (v-cath)1. In an infected insect, chitinase and cathepsin facilitate host cuticle breakdown and tissue liquefaction at the very late stages of infection, so releasing the virus to infect more hosts.

Confocal and electron microscopy observations of insect cells infected with AcMNPV have shown that chitinase is targeted to the endoplasmic reticulum (ER), where it is densely packed in a para-crystalline array, blocking and severely compromising the function and efficacy of the secretory pathway1. V-cath accumulates in the ER at early times post-infection as an inactive proenzyme (pro-v-cath) and is then activated by proteolytic cleavage upon cell death, but is sensitive to the cysteine protease inhibitor E-641.

It has optimum activity at pH 5.0-5.5, although it also shows measurable activity up to pH 7.01. Chitinase may act as a chaperone for the proper folding of pro-v-cath in the ER1. Together these enzymes compete with the recombinant protein for limiting cellular resources, putting a huge burden on the protein translocational machinery1. As a protease, v-cath will also degrade susceptible recombinant proteins, particularly in the later stages of infection when the polh promoter is most active.  

The deletion of both chiA and v-cath from flashBACGOLD™ has improved the efficacy of the secretory pathway and resulted in a greatly enhanced yield of recombinant proteins that are secreted or membrane targeted (in comparison to recombinant viruses that encode chiA and v-cath). Results also show a significant reduction in degradation of protease-sensitive targets and increased production and stability of some intra-cellular proteins1.

Note 1: Contact us for information on the references cited
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flashBACULTRA™
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 flashBACULTRA™ has taken this technology a step further, by the removal of three more virus genes (p10, p74 and p26) from the flashBACULTRA™ genome.

p10 is a 10 kDa protein, expressed concurrently with polyhedrin (polh) late in infection and is nonessential in cell culture. Both p10 and polh promoters share a 12-nucleotide consensus sequence containing the transcription initiation ATAAG motif. p10 is activated a few hours before polh and has been demonstrated to compete with polh at a transcriptional level. However, inhibition and deletion of the p10 promoter has been shown to result in increased polh-controlled protein production and polh mRNA levels. p10 also associates with occlusion bodies (OBs) and is believed to mediate nuclear disintegration late in infection as its disruption has been shown to prevent OB release. Recent work has also shown that it forms extensive cytoskeleton-associated or cytoskeletal-like structures in the nucleus and cytoplasm, potentially de-stabilizing the cells cytoskeleton and further depleting cellular resources.

Deletion of p10 increases polh activity providing more recombinant protein, increases nuclear and cellular stability, ensuring a longer time frame for protein expression and removes a major competitor for limiting cellular resources.

P74 is non-essential in cell culture but is essential for oral infectivity of occlusion-derived virus (ODV) in the host where it plays a role in midgut attachment and fusion. Deletion of p74 has been shown to have no effect on virus production in vitro.

Deletion of p74 further increases the biosafety profile of recombinant baculoviruses in the environment, making them unable to traverse the insect gut wall.

P26 is an early gene that codes for a 240-amino acid polypeptide of unknown function and has the same 5′ terminus as p10. Deletion of the 3’-end of p26 and fusion to lacZ or p10 have previously been shown to have no effect on virus replication in vitro.

Deletion of p10, p74 and p26 removes an unnecessary genetic burden from the recombinant virus genome, providing a more efficient baculovirus expression vector.
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