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Introduction of Bac-to-Bac Expression System

2020-12-01
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Introduction of Bac-to-Bac System

The baculovirus-insect cell system is a binary system consisting of two parts. The first part is the baculovirus expression vector, which is an insect virus whose function is obviously to introduce the foreign gene encoding the target protein into the host cell. Another function of baculovirus expression vectors is to provide the required transcription complex for the transcription of target genes under the control of late or very late promoters. The second part is the host, usually a lepidopteran insect cell line, but sometimes a lepidopteran insect.

bac to bac expression

Baculovirus Expression Vector System

Baculovirus expression vector system (BEVS) is a power system to produce a variety of prokaryotic and eukaryotic protein.  It is also an efficient tool for preparing vaccines.

The system could effectively produce high yield target protein with proper folding and post-translation modifications in an intracellular or secretion expression means.

Medicilon’s Baculovirus Expression Vector System Services

Medicilon researchers establish a well-developed baculovirus-insect cell expression services platform. We provide the expression and purification services on preparation of recombinant baculovirus and recombinant protein. We have a good track record in producing kinase and recombinant protein complexes.

Service:

  1. Generation of recombinant Bacmid DNA

  2. Preparation of recombinant baculovirus

  3. Titration of baculovirus

  4. Protein expression verification and optimization

  5. Small scale expression and purification of recombinant protein in insect cell

  6. Scale up expression and purification of recombinant protein in insect cell

StepsItemsTimelineComments
1Generation of recombinant Bacmid DNA3 weeksDifferent affinity tag: GST tag, His tag, Flag tag…..
2Preparation of recombinant baculovirus2 weeks
3Titration of baculovirus2-3 daysQuantitative-PCR or based on gp64 antibody
4Protein expression verification and optimization2 weeksMOIs, PIs or different cell lines
5Small scale expression and purification2-4 weeks500 ml-2L expression in flask
6Scale up expression and purificationTBDCulture in flask or fermentor

Advantages of Baculovirus Vectors

(1) Easy to operate. Baculovirus has a small genome, simple molecular biological characteristics, and a variety of restriction endonuclease sites on the genome.

(2) Large foreign genes can be accommodated. Baculovirus’s virus particles are rod-shaped and have relatively strong plasticity to accommodate foreign genes. In theory, it can accommodate any foreign genes.

(3) It can express foreign genes efficiently. In baculovirus genes, p10 gene and polyhedrin gene are both extremely late genes, and their promoters can efficiently express the target protein.

(4) The target protein can be modified after expression. The host of baculovirus is insect cells, and the target protein can be processed and modified using the post-translational modification system of insect cells.

(5) High security. Baculovirus expression vectors are very specific and can only replicate in insect cells.

(6) Low cost. Compared with mammalian cells, insect cultured cell lines are relatively easier to grow, and insect cells can be cultured in suspension to produce large amounts of the target protein.

Baculovirus-insect Cell Expression System Design

(1) Using the protein sequence translated from the target gene sequence as the source template, a set of gene sequences more suitable for SF9 cell line are optimized through codons.
(2) According to the results of the target gene sequence analysis, see if the target gene contains a signal peptide. If there is no signal peptide, the GP67 signal sequence needs to be added to the N-terminus of the protein sequence to help the protein to be secreted and expressed outside the cell during gene recombination. Adding 6XHis-Tag to the end facilitates the detection and affinity purification of recombinant proteins.
According to the above design ideas, the pFast-bac1-target-gene expression plasmid was constructed by gene synthesis. After transformation, the recombinant Bacmid was obtained by the blue-and-white screening method. After PCR identification, it was transfected into sf9 cells to obtain P1 generation virus and P2 generation. virus. Infect 200ml small test expression, detect protein expression by western blot, confirm the protein expression, and then obtain the target protein with more than 80% purity by Ni Focurose 6FF (TED) affinity purification.

Related Articles:

Baculovirus Expression System

Baculovirus Expression Vector System

Introduction of Bac-to-Bac System

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