Recombinant expression of proteins and their complexes using the baculovirus/insect cell expression system (BEVS)
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The main steps for production in insect cells using the baculovirus expression system are insertingf the gene(s) of interest into a baculoviral transfer vector of choice, followed by isolation of the recombinant baculovirus, amplification of viral stocks and infection of exponentially growing insect cell cultures for protein production.
Construction of transfer vectors will be mostly performed in the user's laboratory or on site, depending on the complexity of projects and the expertise of users. Quality controlled reagents, standard operating procedures in form of stream-lined protocols and expert subervision are provided by the host infrastructure. Assistance in planning and design of the experiment isprovided upon request by the user. Generation of the recombinant viruses, expression screening and medium-scale production is performed at the host infrastructure. The procedure entails from several days (e.g. cloning at user's laboratory and basic expression screening) to two/three weeks (e.g. multigene cloning and complex expression at an Instruct centre). Users return to their home laboratories with reagents including constructs, viruses, cell pellets and, in many cases, with purified samples.
There are several ways to generate a recombinant baculovirus. The currently most commonly used method relies on Tn7 mediated transposition of genes from a transfer plasmid to a baculovirus present as an artifical bhacterial chromosome (bacmid) in customized E. coli cells (Bac-to-Bac, MultiBac systems). The resulting recombinant baculovirus is then obtained from an E.coli culture by alkaline lysis and used to transfect insect cell cultures. Alternatively, homologous recombination can be utilized between a purified baculoviral genome and a different transfer plasmid containing the gene(s) of choice in adddition to defined homology regions to the baculovirus genome (BaculoGold, BacVector series, FlashBac). Here, transfer plasmid and baculoviral genome are together applied to insect cells using a transfection reagent. For both approaches, an array of plasmids and numerous cloning strategies are available, most of which are compatible with parallelised and robotised strategies.
A variety of baculovirues and cell lines (Sf9, Sf21, Hi5) are available at the host platforms. Expression experiments can be carried out in adherent or suspension culture, at different scales (up to several Liters in suspension culture). Adherent cultures are recommended for parallel expression screening (typically in a 6 well plate), or protein-protein interaction studies by co-infection with multiple viruses (typically using T175 for flasks). Medium to large scale expression is typically performed in suspension cultures (0.5-6 liters for standard production) for production of proteins and protein complexes for functional, biophysical and structural studies.