FlashBAC Expression Approach

In order to shorten the expression period, baculovirus DNA backbone can be co-transferred with transfer vectors (which carry the gene of interest) to directly generate recombinant virus by intracellular homologous recombination. However, the recombination rate can be frustrated sometimes because the non-recombinant DNA can still direct virus formation. The strategy which inactivate baculovirus backbone by replacing essential elements and reactive the virus replication function by co-transfecting backbone and transfer vector is called rescue strategy. This strategy is commonly used to eliminated non-recombinant parental virus background.

There are many expression approaches that refer to this strategy, but none of them can produce 100% positive vector (such as BacPAK6). Customers who is looking for a 0% negative expression approach please consider the FlashBAC approach Creative BioMart provides. Benefit from the brilliant rescue strategy and the baculovirus virus backbone manipulation, the recombinant rate between transfer vector and virus backbone can reach 100%. As a result, this approach omits the steps of plaque screening thus significantly reduces the period of protein expression.

Technology Description

This system consists of an AcMNPV genome as well as a transfer vector. The major manipulations are carried out on the AcMNPV genome.

First of all, the essential gene ORF 1629 is partly delated. Consequently, the non-recombinant virus gene backbone cannot self-replicate when transfected into insect cell
Secondly, polh coding sequence is replaced with a bacterial artificial chromosome (BAC) at the polh locus. BAC allows the virus genome to be maintained in bacterial cells as a bacmid.

All of the deletion can be found in the transfer vector. The transfer vector contains

A complete ORF 1629, which is essential for DNA replication
A polh coding sequence, which is related to the strong promotor
A sequence of gene of interest, which codes the target protein

When the co-transfection is conducted, the ORF 1629 is restored in the insect cells to allow the recombinant virus to replicate. In addition, the BAC fragment is replaced by the target gene and the polh coding sequence. The non-recombinant viral genome cannot survive thus can be directly used for virus assembly, proliferation, protein expression and other follow-up steps without plaque screening.

Further Modifications of FlashBAC Expression Approach

Name	Modification	Feature
FlashBAC	Backbone virus DNA chiA delectation	A. Prevent production of virus chitinase. B. Improved membrane and secreted protein production.
FlashBAC GOLD	Backbone virus DNA chiA and v-cath delectation	A. Prevent production of virus chitinase and cathepsin. B. Improved membrane and secreted protein production. C. Prevent susceptible protein degradation.
FlashBAC ULTRA	Backbone virus DNA chiA, v-cath and p10/p26/p74 delectation	A. Prevent production of virus chitinase and cathepsin. B. Improved membrane and secreted protein production. C. Prevent susceptible protein degradation. D. Extend protein production times in T. ni Hi5 cells. E. Reduces the metabolic burden on the cell
FlashBAC PRIME	No gene deletions.

Why Choose FlashBAC Expression Approach

FlashBAC approach is easy operation.
Simplified process——does not require plaque-purification.
Amenable to making high throughput expression——manually or using a robot in 24-well plates.
Ideal for secreted or membrane targeted proteins.
Ideal for difficult protein production.

Service Details of FlashBAC Approach

Here is the service process, which is very flexible. Customers can choose to start from or stop at any step they want. For more technical information or enquiry, please feel free to contact us.

Step		Service Description	Timeline	Deliverables
1	System Selection （Optional）	Select transfer vector Select host insect cell line	——	——
2	Prepare Target Gene	Target gene sequence design and codon optimization Gene synthesis Subclone the interest sequence to the donor plasmid	< 1 week	Enzymatic / Sequencing validation report.
3	Prepare Transfer Vector	Subclone the PCR product to the transfer vector Verify correct construct Extract recombinant vector	< 1 week	Recombinant transfer vector Analyze report
4	Recombinant Baculovirus Production	Co-transfect insect cell with transfer vector and viral DNA Co-transfection of positive control Harvest P0 virus stock	1 week	Recombinant virus Recombinant rate report
5	Recombinant Virus Evaluation & Amplification	Amplify virus from P0 to P1 to P2 Virus titration	1 week	P1 and P2 virus stock Virus titration report
6	Test Protein Expression	Protein expression Target protein evaluation	1 week	Protein product Protein activity report
7	Large-Scale Expression & Purification (1-10L)	Culture amplification Western Blot Purification	1-3 weeks	Purified Protein QC report

Creative BioMart has more than 10 years of experience in protein expression and is competent to satisfy various protein expression desire. We are expert in protein expression (intracellular or secretory), signal peptide selection, insect cell selection and culture. Choosing our comprehensive service, customers only need to provide the coding sequence of your target protein. We will provide customizes service from codon optimization to large-scale expression and purification.

Please note that all products/services provided are for research use only. Not intended for any clinical use.