BacTen baculovirus is an original and powerful tool for the generation of p10 promoter based expression vectors. The BacTen expression system has been designed to permit efficient expression of foreign proteins in insect cells. In the BacTen baculovirus, the polyhedrin promoter has been deleted. The foreign genes are expressed from a single strong late promoter construct in the absence of the highly produced viral proteins P10 and polyhedrin.

The BacTen system takes advantage of some important features :

As the polyhedrin gene expression is abolished, the foreign gene expression driven by the p10 promoter is placed in a favourable context for optimal production of the recombinant protein.

The p10 promoter has been shown to be activated earlier in infection time courses than the polyhedrin promoter allowing for the earlier initiation of expression of some recombinant proteins.

In BacTen based recombinants, no P10 polypeptide is produced. This delays the lysis of infected cells and allows for a greater cumulative expression of recombinant proteins.

Steps for BacTen recombinant construction

1. The gene of interest is inserted downstream of the p10 promoter in a transfer vector (see vector maps).
2. Sf9 cells are co-transfected with BacTen linearized DNA and the transfer vector bearing the gene of interest.
3. The supernatant from the co-transfection is collected. At this stage, cell and or supernatant extracts may be analysed to detect recombinant protein production.
4. Recombinant viral clones are isolated from the co-transfection supernatant, using the occlusion body-negative phenotype to select recombinants.
5. Amplification of the recombinant viruses.
6. Analysis of culture extracts to detect the recombinant protein and compare the isolated viral clones.
7. Cell infection with a selected virus to generate a high titre stock that can serve for small or large-scale productions of the recombinant protein

More information Download the Baculovirus Services Brochure (pdf)

The BacTen system

Relaying on our extensive knowledge of molecular biology technologies, we developped a full range of vectors.

QBI-pTen12 and QBI-pTen 21 - Standard BacTen expression vector for convenient cDNA cloning
QBI-pTenACE - Signal peptide for secretion of recombinant polypeptide
QBI-pTenTwin - Simultaneous co-expression of two recombinant polypeptides
QBI-pTenHis - 6 x His-Tagged recombinant proteins for simple purification

QBI-pTen12 and QBI-pTen 21
Standard BacTen expression vector with polylinker for convenient cDNA cloning

These transfer vectors contain viral sequences flanking the p10 gene to permit homologous recombination with linearized BacTen DNA, the p10 promoter and a series of unique restriction sites for cloning the sequences to be expressed. pQBI-pTen12 and QBI-pTen21 are identical except that their multiple cloning sites (MCS) are inverted. These plasmids also carry the ampicillin resistance gene for selective growth in Escherichia coli.

QBI-pTenACE
Signal peptide for secretion of recombinant polypeptide

Vector QBI-pTenACE contains a sequence encoding the signal peptide (SP) of Drosophila melanogaster acetylcholinesterase (an insect protein) downstream of the p10 promoter. This protein has been shown to be efficiently secreted and processed when produced in Spodoptera frugiperda cells (Chaabihi et al., 1994). The pMultiple cloning site is positioned to allow in-frame fusion of foreign sequences with the SP sequence. Upon transfer into BacTen DNA, the produced chimeric protein should be targeted to the secretory pathway of insect cells. Usually, signal peptides of naturally secreted proteins are functional when these proteins are produced in insect cells. However, in a few cases, the natural leader sequences fail to function correctly. In these cases, replacement of the natural leader sequence with the ACE sequence may restore efficient secretion of the recombinant protein.

QBI-pTenTwin Double expression cassette for simultaneous co-expression of two recombinant polypeptides
Vector QBI-pTenTwin is designed for use with BacTen DNA when co-expression of two genes is desired. It contains a p10 promoter and a synthetic p10-like promoter, which are placed end-to-end. Unique restriction sites are placed downstream of each promoter. Transcription directed by the synthetic promoter will stop at the original p10 transcription stop signal. For the p10 promoter, a thymidine kinase-gene signal (TKPA) has been introduced to stop transcription and permit polyadenylation of mRNAs. For genes to be expressed concomitantly, each gene is introduced downstream of a promoter. The standard procedures (co-transfection, isolation of recombinant viruses, etc.) are then followed.

Sequence of multiple cloning sites

Contact our Experts to discuss your needs.

North America: 800-424-6101
Europe: 33 388 675 423

Email:  More Information