BaculoDirect System

The process of site-specific transposition can be time-consuming. The BaculoDirect Baculovirus Expression System uses Gateway technology to insert the gene of interest into the baculovirus genome in vitro, eliminating the section of transforming bacteria, bacmid extraction, or co-transfection. Through the Gateway recombination reaction, the recombinant baculovirus genome needed for transfection can be obtained within one hour, dramatically shortens the protein expression period.

How Baculovirus BEVS Works

Gateway is based on the integration of phage DNA into the host bacterial genome. Under the action of some integration factors, reversible recombination can occur between highly conserved att sites.

In the BaculoDirect system, the gene of interest is flanked with attL segment to firm an entry clone. Meanwhile, the baculovirus genome (BaculoDirect Linear DNA) is modified to contain attR sites. After a one-hour LR reaction triggered by LR Clonase II, the gene of interest is recombined from the entry clone to the baculovirus genome. The resulting mix is then be used to infect insect cells.

Why choose BaculoDirect BEVS

BaculoDirect allows rapid generation of recombinant virus.
Suitable for production of high titer stock.
LR reaction can be completed within one hour.
No need for transforming bacteria, bacmid extraction, or co-transfection.
Produce high level epitope tagged recombinant protein which is easy for detected and purified.
No need for plaque purification.
Cost saving. The LR recombination process does not require endonucleases.
Once built, an entry clone can be used multiple times
The ORF and direction of DNA fragment remain unchanged during LR recombination

Our service for BaculoDirect BEVS

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 entry vector Select host insect cell line (Sf9, Sf21, High Five)	——	——
2	Gene Synthesis	Sequence design and codon optimization Gene synthesis	>weeks	Gene of interest Sequencing report
3	Prepare Entry Clone	TOPO clone the gene of interest to entry vector	5 mins	Entry clone that contains target gene
4	Conduct LR Recombinant Reaction	Mix baculovirus linear DNA and entry clone with LR Clonase Transfect the insect cells with recombinant DNA	1 hour	Recombinant baculovirus DNA
5	Recombinant Baculovirus Production	Produce P1 Virus stock (low titer), P2 Virus stock (high titer), P1, P2 Western Blot and titer assay	1 weeks	Recombinant virus suspension WB report Titer test report
6	Small-Scale Expression & Purification	P2 virus transfect insect cell Protein purification SDS-PAGE, Western Blot	1.5 weeks	Target protein sample Quantitative test report
7	Optional Services	System optimization Secondary purification Tag removal Activity assay	1-2 weeks	Optimization solution Protein product Protein activity report
8	Large-Scale Expression & Purification (1-10L)	Culture amplification Western Blot Purification	1-3 weeks	Purified Protein QC report

Entry Vector and Insect Cell Line

In order to optimize the expression approach, entry vectors and insect with unique advantage are available at Creative BioMart.

Entry Vector

Entry Vector	Use
pENTR/ TEV /D-TOPO	Directional TOPO cloning 5’ TEV sequence for N-terminal tag removal
pENTR/ D-TOPO	Directional TOPO cloning
pENTR/ SD /D-TOPO	Directional TOPO cloning Vector includes the Shine-Dalgarno sequence for E. coli expression-ready entry clone

Insect Cell Line

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
High Five (Trichoplusia ni)	High yield of secreted protein, shorter culture period	Secreted 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.