Protein Expression in Insect Cells Via Baculovirus
Allele Biotechnology has been a leader in the Baculovirus field since 2008 through its acquisition of Orbigen Inc., a pioneer in the baculovirus field. Over the years we have developed numerous transfer plasmids, enhanced insect cell lines, the BaculoBank and groundbreaking technologies to aid protein expression, examples of this being the first functional insect IRES in baculovirus-mediated gene expression and booster viruses. In most cases, using baculovirus system correctly expresses proteins that are found to be insoluble in bacteria, and in sufficiently large quantities that cannot be meet in mammalian expression. A typical protein expression/purification project using baculovirus expression system starting from cDNA sequence data will take ~ 2 months to complete; this is currently the fastest method to obtain mg amounts of soluble protein. Over the years, we have completed hundreds of baculovirus expression/purification projects.
Below we have outlined the various steps in a typical baculovirus custom service project for protein expression. As each custom project is unique, users can select the steps they would like and omit the others to tailor to their specific project. Please contact us (firstname.lastname@example.org) for technical questions and quotation.
Subcloning into Recombinant Baculovirus Transfer Vector
- Design/construct the gene of interest into the elected recombinant Baculovirus transfer vector.
- Verify the subcloned gene by restriction enzyme digest and sequencing.
- Prepare the recombinant vector DNA suitable for co-transfection with our proprietary baculovirus DNA.
- 5 ug of plasmid containing gene of interest plus complete vector sequence in electronic format.
- Target protein/cDNA sequence (codon optimization and gene synthesis)
- 5 ug of ready-to-use recombinant Baculovirus transfer plasmid containing the gene of interest
- Verified sequence of baculovirus transfer plasmid containing the gene of interest.
- 2 weeks: If insert > 2kb, one week additional per 1kb
- $999: If insert > 2kb, $399 additional per 1kb
Generation and Evaluation of Baculovirus Recombinants
- Cotransfect recombinant Baculovirus transfer vector containing gene of interest with 3 different proprietary linearized Baculovirus DNAs using BaculoShuttle transfection reagent.
- Identify and amplify three recombinant constructs.
- Select the best recombinant baculovirus construct for the protein expression.
- Amplify three rounds to generate a high titer virus stock (>1e8 pfu/ml)
- Titrate high titer stock using end point dilution
- 10 ug of ready to use recombinant Baculovirus transfer plasmid containing the gene of interest.
- Three cell pellets or baculovirus-conditioned insect cell media for evaluating Baculovirus-mediated protein expression
- 30ml of ready-to-use baculovirus high titer stock (>1e8 opfu/ml)
- 2 - 4 Weeks
Amplification of Recombinant Baculovirus Stock
- Amplify low titer recombinant Baculovirus to high titer stock (>1e8 pfu/ml).
- Titrate high-titer stock by end point dilution
- 5 ml of high-titer (>1e8 pfu) recombinant Baculovirus stock.
- 500 ml of high-titer recombinant Baculovirus stock solution.
- 2 - 3 Weeks
- $599 per 500ml
Baculovirus Expression Optimization Service
- Prepare/grow log phase insect cultures
- Grow adherent insect cultures on Phoenix Tissue Culture dishes.
- Infect three insect cell lines (Sf21, Sf9, and T.ni)
- Infect at low and high multiplicity of infection (MOI)
- Harvest cells and Baculovirus-conditioned cell media at 48hr and 72hr post-infection
- At least 5-10 ml of recombinant Baculovirus stock.
- 18 pairs of cell pellets and virus conditioned cell media for evaluation of protein expression
- 2 - 4 Days
Protein Production Service
- Prepare high titer recombinant Baculovirus stock for protein production.
- Prepare adherent cell culture using Phoenix Tissue Culture dishes (~34 large 150x25mm dish/liter).
- Infect cells under select conditions.
- Harvest/process cells or conditioned media at selected time postinfection.
- High titer recombinant Baculovirus (25 ml per liter of protein production desired).
- Cell pellets or conditioned cell media containing target protein.
- 2~3 weeks (1~5 Liters)
- 4~5 weeks (5~20 Liters)
- 8~10 weeks (20 Liters and above)
- For multiple liters please ask for quotation
Protein Extraction & Purification
- Process cell pellet or cell media from 1 liter of insect using our proprietary reagents and SOP.
- Purify either under native or denatured conditions using appropriate affinity resin for 6xHis or GST-tagged recombinant proteins.
- SDS PAGE analysis
- Dialyze/buffer exchange purified protein in storage buffer.
- Cell pellet or baculovirus-conditioned cell media containing the target protein.
- Purified protein in standard or customer-specified storage buffer.
- SDS-PAGE data
- 2 - 3 Weeks
- $1,500 for 1 Liter
- Inquire for larger quantities