BacPAK6 System

In order to shorten the expression period, circular baculovirus DNA 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.

Creative BioMart provides the BacPAK6 expression system to facilitate the expression vector recombination rate and the selection of the positive vector (Figure 1). A rescue strategy is adopted to inhibit the replication of non-recombinant virus. Benefit from the brilliant rescue strategy, over 95% of the recombination rate can be promised at Creative BioMart.

Creative BioMart also provide a In-Fusion Ready BacPAK Vector which is designed to attach a 6xHN tag at N- or C-terminal of protein. The tagged protein can be easily purified using TALON resin.

BacPAK6 System

This system consists of a liner AcMNPV genome (Liner BacPAK6 DNA) as well as a transfer vector (pBacPAK). The essential ORF 1629 segments on virus DNA is removed by Bsu36I enzyme digestion. It is unable to replicate unless co-transferred with the transfer vector which contains a complete ORF 1629. Creative BioMart’s BacPAK6 protein expression system exhibits the feature of:

Allows the direct cloning of PCR product into the transfer vector.
The BacPAK6 Viral DNA is prelinearized, there is no need for addition recombinant operation
Enhanced recombinant efficiency – over 95%
Simplified recombinant viruses screening

Figure 1. Expression Approach of BacPAK6 System

In-Fusion Ready BacPAK

An In-Fusion Ready BacPAK Vector is introduced to replace the conventional pBacPAK. A coding sequence of 6xHN (polyhistidine) is attached at the C- or N-terminal of the transfer vector. The recombinant protein product with 6xHN tag can be easily purified by using TALON resin. The In-Fusion Ready BacPAK Vector exhibits the feature of:

Allows the direct cloning of PCR product into the transfer vector.
Prelinearized BacPAK6 Viral DNA and transfer vector, there is no need for restriction enzyme pre digestion.
Enhanced recombinant efficiency – over 95%.
Simplified recombinant viruses screening.

Service Details of BacPAK6 System

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 (pBacPAK vector/ in-fusion ready BacPAK vector) Select host insect cell line (Sf9, Sf21, High Five)	——	——
2	Prepare Target Gene	Target gene sequence design and codon optimization Gene synthesis Subclone the interest sequence to the donor plasmid	< 1week	Enzymatic / Sequencing validation report.
3	Prepare Transfer Vector	Subclone the PCR product to the transfer vector Verify correct construct Extract recombinant vector	< 1week	Recombinant transfer vector Analyze report
4	Recombinant Baculovirus Production	Co-transfect insect cell with transfer vector and linear viral DNA Co-transfection of positive control Plaque assay and positive recombinant virus confirmation	1 weeks	Recombinant virus Recombinant rate report
5	Recombinant Virus Evaluation & Amplification	Prepare and assay of P1 virus Prepare P2 virus Virus titration	1.5 weeks	P1 and P2 virus stock Virus titration report
6	Optional Services	System optimization Secondary purification Tag removal Activity assay	1-2 weeks	Optimization solution Protein product Protein activity report
7	Large-Scale Expression & Purification (1-10L)	Culture amplification Western Blot Purification	1-3 weeks	Purified Protein QC report

Transfer Vector and Insect Cell Line

In order to meet the various expression requirement, pBacPAK transfer vectors and insect cells with unique advantage are available at Creative BioMart.

Transfer Vector

Donor Vector	Use
pBacPAK8/9	Contains a strong polyhedrin prompter. Suitable for high-level protein expression. The sequence direction of MCS of pBacPAK8 and 9 is opposite
pBacPAK8-Nterm 6xHN	Contains a strong polyhedrin prompter. Suitable for high-level protein expression. Frame the IMCA tag at N-terminal of the PCR product Suitable for TALON resin
pBacPAK8-Cterm 6xHN	Contains a strong polyhedrin prompter. Suitable for high-level protein expression. Frame the IMCA tag at C-terminal of the PCR product Suitable for TALON resin
pBacPAK8-GUS	Positive control of pBacPAK8/9 Used to monitor transfection efficiency and virus production
pBacPAK8-Nterm 6xHN-GUS	Positive control of pBacPAK8-N/Cterm 6xHN Used to monitor transfection efficiency and virus production

Insect Cell Line for BacPAK

Insect Cell	Advantage	Use
Sf9 (Spodoptera frugiperda)	Well-characterized host cell, good for plaquing	All general types of recombinant baculovirus production and protein expression
Sf21 (Spodoptera frugiperda)	High yield of intracellular protein	Intracellular recombinant baculovirus production and protein expression

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.