Fig. 1
Overview of the FlexiBAC baculovirus expression system. a Recombination between a SbfI-linearized defective viral backbone (“DefBac”) and a shuttle vector (“pOCC”, which contains the target gene of interest) creates a viral genome capable of producing infectious virus. Recombination occurs between complementary lef2 and AcORF1629 truncations located on the pOCC vector and the DefBac viral backbone. Recombination generates full-length versions of lef2 and AcORF1629 genes, which are needed for baculovirus production. No virus is produced without proper recombination, thus eliminating the need for post-production screening for recombinant virus. The DefBac viral backbone also includes deletions in the genes encoding cathepsin and chitinase. A second version of DefBac, called DefBacFur+, expresses the convertase furin. b Each pOCC shuttle vector contains a modular expression cassette that allows insertion and swapping of the gene of interest, N-terminal tags, and C-terminal tags using classic cloning techniques (restriction sites are shown with arrowheads). A gene encoding the ccdB toxin selects against plasmids lacking the gene of interest. c pOCC shuttle plasmids encode a variety of tags that can be appended to the target protein of interest. Tags are easily combined and swapped to create customizable N-terminal or C-terminal fusions. Descriptions of each tag and the available combinations (143) are shown in Table 1 (see Additional file 1) and available upon request. The most commonly used plasmids (52) are shown in Table 2 (see Additional file 2) and are available at www.addgene.org. d Timeline for production of recombinant baculovirus using the FlexiBAC system. On day 1, the user transfects Sf9 insect cells with pOCC shuttle vector (with target gene of interest) and linearized DefBac DNA. Within some insect cells, pOCC and DefBac will recombine and this event generates an infectious baculovirus that propagates throughout the culture. On day 5, the user collects the conditioned media (which contains released baculovirus) and uses it to infect fresh Sf9 insect cells. On day 10, the user uses the conditioned media again to infect fresh Sf9 cells. On day 13, the user harvests the infected Sf9 cells containing the target protein of interest (represented by green, GFP+ cells). For secreted protein targets the conditioned medium is collected after this phase