Baculovirus Expression System (5 reactions) (GWB-7676BB)



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The Baculovirus Expression System has been specifically designed to remove the need to separate recombinant virus from parental virus by plaquepurification or any other means. The production of recombinant virus has been reduced to a one-step procedure in insect cellsand is thus fully amenable to high throughput and automated production systems.

This system utilizes an AcMNPV genome that lacks part of an essential gene (ORF 1629) and contains a bacterial artificial chromosome (BAC) at the polyhedrin gene locus, replacing the polyhedrin coding region. The essential gene deletion prevents virus replication within insect cells but the BAC allows the viral DNA to be maintained and propagated, asa circular genome, within bacterial cells. Circular viral DNA is then isolated from the bacterial cells and purified. This is the co-transfection factor.

A recombinant baculovirus is produced by simply transfecting insect cells with co-transfection factor and a transfer vector containing ‘the gene under investigation’. Homologous recombination within the insect cells (1) restores the function of the essential gene allowing the virus DNA to replicate and produce virus particles and (2) simultaneously inserts ‘the gene underinvestigation’ under the control of the polyhedrin gene promoter and removes the BAC sequence. The recombinant virus genome, with the restored essential gene, replicates to produceBV that can be harvested from the culture medium of the transfected insect cells (and forms a seed stock of recombinant virus). As it is not possible for non-recombinant virus to replicate there is no need for any selection system.

This one-step procedure greatly facilitates the high throughputproduction 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.

The Baculovirus Expression System is back compatible with all baculovirustransfer vectors based on homologous recombination in insect cells at the polyhedrin gene locus. This includes vectors using the polyhedrin promoter, dual, triple and quadruple expression vectors and those that use other gene promoters such as p10, ie1 etc. Examples include pBacPAK8/9, pAcUW31 and pBacPAK-His1/2/3 (BD Biosciences Clontech) but not vectors such as pFastBac™, which are designed for site-specific transposition in E. coli using the Bac-to-Bac® system (GibCoBRL).

The Baculovirus Expression System also maximises protein secretion and membrane protein targeting. Baculovirus genomes contain several auxillary genes, which are non-essential for replication in insect cell culture. One of these is chitinase (chiA), whichencodes an enzyme with exo- and endochitinase activity. In an infected insect, chitinase (together with cathepsin) facilitates 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, severely compromising the function and efficacy of the secretory pathway. Deletion of chiA from flashBAC has improved the efficacy of the secretory pathwayand 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).

Advantages of the Baculovirus Expression System
• Simple to use
• One step production of recombinant virus in insect cells
• No steps needed to purify recombinant virus
• Amenable to high throughput and automated systems
• Maximises production of secreted and membrane-targeted proteins
• Back-compatible with a huge range of transfer vectors.

Additional Information

Name Baculovirus Expression System (5 reactions) (GWB-7676BB)
Kit Component Co-transfection Factor - Use 100ng [5ul] per co-transfection 20ng/ul.
Positive-control vector DNA - Use 500ng [5ul] per co-transfection 100ng/ul.
Application To be provided by the user:
Transfection Reagent - reagents tested and found to be successful: baculoFECTIN II© (OET), Lipofectin© (Invitrogen), FuGENE 6 (Promega), GeneJuice© (Novagen)
Transfection media (e.g. Grace's Insect Basal Medium or TC100)
35mm tissue culture treated dishes seeded with Sf21 (1.4 x 106 cells/2mls), or Sf9 (1 x 106 cells/2mls) per dish
Prepare 2 hours before transfection
Sterile baculovirus transfer vector DNA containing gene of interest (500ng per co-transfection)

Procedure: (as recommended with use of baculFECTIN II. Other reagents may differ.)
Prepare DNA transfection complexes
1. Warm baculoFECTIN II to RT and vortex gently.
2. Place 100ul transfection medium in a sterile tube.
3. Add 5ul (100ng) co-transfection factor. Tap tube to mix.
4. Add 5ul (500ng) plasmid transfer vector. Tap to mix.
5. Add 1.2ul of baculFECTIN II. Tap to mix.
6. Incubate at RT for 15-20 minutes.
Add DNA complexes to cells.
1. Remove 1mL medium from each dish required.
2. Add baculoFECTIN II:DNA complex mixture drop-wise to different areas of the dish.
3. Incubate 16-24 hours.
4. Add 1mL fresh medium to each culture dish.
5. Incubate for 4 days at 28C (5 days from start of co-transfection).
Harvest P0 virus stock
1. Aspirate medium from dishes and remove cells at 3000 rpm for 10 minutes.
2. Amplify virus stock.
Reconstitution and Storage Co-transfection Factor - Store at 4C
Positive-control vector DNA - Store at -20C
Stable for 1 year from date of receipt when properly stored and handled.
Datasheets/Manuals Printable datasheet for GWB-7676BB
Intended Use Research Use Only