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The Bac-to-Bac insect baculovirus expression system allows for the rapid and efficient preparation of recombinant baculoviruses that rely on a site-specific transposon to recombine an intact expression element onto a baculovirus shuttle plasmid (bacmid). The system contains the following main components. The pFastBac vector-contains a baculovirus-specific promoter that forms a complete expression element after insertion of the target gene downstream of it. This expression element is then inserted into the shuttle plasmid as a transposon. DH10Bac strain-contains a baculovirus shuttle plasmid (bacmid), and a helper plasmid. The helper plasmid expresses transposase and helps to complete the transposition process.
Traditional baculovirus systems require purification and amplification of an initial low-titer viral supernatant. In contrast, the Bac-to-Bac baculovirus expression system is faster and easier because the technology relies on generation of recombinant baculovirus by site-specific transposition in E. coli rather than homologous recombination in insect cells, which often results in low titers. Lifeasible provides protein expression services with Bac-to-Bac baculovirus expression system. The details of the services we provide are listed below.
| Service content | Detail |
| Full gene synthesis | Analyze gene sequences, optimize codons, synthesize target genes, and provide vectors. |
| Construction of recombinant plasmids | Construct recombinant donor plasmid 'pfastbacl-target'(target represents target gene) with suitable vector and suitable tag (eg: His, GST, Fc, SUMO). |
| Preparation of recombinant baculovirus plasmids | Transformation of recombinant donor plasmid into DH10bac receptor cells, blue and white spot screening of positive clones and repeated identification using PCR, and finally alkaline lysis of small extracts of recombinant baculovirus plasmids. |
| Cell transfection | Resuscitate and transfect suspension-cultured SF21 cells with recombinant bacilli into SF21 cells using chemical transfection reagents to prepare P1 generation recombinant baculovirus. |
| Expression of recombinant proteins | Preparation of P2 generation viruses using P1 generation baculovirus, use of P2 generation viruses to infiltrate large-scale cultured SF21 cells to express recombinant proteins. |
| Isolation and purification of recombinant proteins and QC assay | The recombinant proteins are separated and purified by suitable chromatographic methods (eg: affinity, ion, size exclusion, etc.) and ultrafiltrated into customer-specified buffers (PBS or Tris-HCl is chosen as the final preservation buffer if there is no special requirement). QC assays include SDS-PAGE for recombinant protein purity, BCA for quantitative recombinant protein concentration, and Western-blot. |
Fig 1. Service flow for the Bac-to-Bac baculovirus expression system.
Lifeasible provides protein expression services with Bac-to-Bac baculovirus expression system. We offer full gene synthesis, construction of recombinant plasmids, preparation of recombinant baculovirus plasmids, cell transfection, and expression of recombinant proteins by our professional team. If you are interested in our services or if you have any questions, please click online inquiry for more detailed information.
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