Protein complexes typically consist of not only proteins but also nucleic acids and small molecules. That being able to generate the complexes is the premise of the structural and functional analysis. However, the conventional polygene expression method is low throughput in subunit gene insertion thus limits the generation of such constructs in parallel. In addition, it has been demonstrated that if the genes or cDNA encoding single complex subunits are combined into one DNA expression vector instead of being introduced into host cells as a single construct, the yield, homogeneity, reproducibility and biological activity of these protein complexes can be improved.
For customers who want to express multisubunit protein complexes, please consider Creative BioMart's biGBac Expression Service. The biGBac approach allows up to 25 subunit gene be assembled into a single baculovirus vector within 2 steps.
The biGBac is a combination of baculovirus Bac to Bac expression and Gibson assembly reaction technology. The working process can be divided into two parts.
Part one is to construct polygenes into one vector. Gibson assembly is adopted to achieve the rapid and efficient recombination of up to 25 individual protein-coding sequences into a single DNA construct in only two sequential reactions. The assembly procedure is shown in the figure 1. This vector plays the same role as the "donor vector" in Bac to Bac expression system, although they do not share the same vector backbone.
Figure 1. biGBac vector assembly and expression procedure
(a). Preparation of subunit gene library
In order to further reduce the time consumption of the whole construction process, a universal subunit gene library was constructed. The individual fragment was cloned into the library vector and amplified by primers when necessary. Click here to view the detail information of the library
(b). Preparation of gene expression cassettes (GECs)
Each individual GEC is decorated with a set of the optimized linker sequences α, β, γ, δ, e, and ω through PCR (see in figure 1 A). Up to 5 subunits can be modified at this step. Use Swal enzyme to digest and release GECs.
(c) Preparation of polygene cassettes (PGCs)
Up to five optimized linker sequences decorated GECs are combined with a linearized vector (pBIG1) to generate PGCs. Use Pmel enzyme to digest and release PGC. The "last" GEC of each pBIG1 also carry the "omega" linker sequence to create an overlap.
(d) Assembly of the PGCs into the ultimate vector
Again, up to five PGCs from different pBIG1 are combined with a linearized vector (pBIG2) to generate the ultimate "donor" vector.
(e)Conduct the Bac to Bac process to produce the target protein complexes
If you are interested in our service, you can check our library information, multi gene vector construction service and corresponding quotation through this link.
A protein complexes expression approach which relies on a range of recombination technology including Cre-LoxP and homing endonuclease (HE).
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 Multi Gene Expression 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. For more assistance, please contact us for help.