Generating large amounts of recombinant protein in transgenic animals is often challenging and has a number of drawbacks compared to cell culture systems. The baculovirus expression vector system (BEVS) uses virus-infected insect cells to produce recombinant proteins to high levels, and these are usually processed in a similar way to the native protein. Interestingly, since the development of the BEVS, the virus most often used ( Autographa californica multi-nucleopolyhedovirus; AcMNPV) has been little altered genetically from its wild-type parental virus. In this study, we modified the AcMNPV genome in an attempt to improve recombinant protein yield, by deleting genes that are non-essential in cell culture. We deleted the p26 , p10 and p74 genes from the virus genome, replacing them with an antibiotic selection cassette, allowing us to isolate recombinants. We screened and identified recombinant viruses by restriction enzyme analysis, PCR and Western blot. Cell viability analysis showed that the deletions did not improve the viability of infected cells, compared to non-deletion viruses. However, expression studies showed that recombinant protein levels for the deletion viruses were significantly higher than the expression levels of non-deletion viruses. These results confirm that there is still great potential for improving the BEVS, further increasing recombinant protein expression yields and stability in insect cells.

Baculovirus-mediated expression has proven to be a robust method of generating recombinant proteins from insect cells. However, generating baculovirus recombinants using traditional techniques is time consuming and tedious. To accelerate the process of insect cell expression, EMD developed a rapid transient transfection-based approach, the InsectDirect System. This approach is well suited for the rapid generation of small to moderate amounts of recombinant protein. For situations that demand the baculovirus approach, we introduced a technology for rapid baculovirus production, BacMagic DNA. To optimize, simplify, and consolidate both systems. we created a single expression vector compatible with both approaches, pIEx/Bac. To direct expression by transient transfection, pIEx/Bac expression vectors feature the homologous region 5 (hr5) enhancer and the immediate early 1 (ie1) promoter from Autographa californica nuclear polyhedrosis virus (AcNPV), a promoter/enhancer combination that uses endogenous insect-cell transcriptional machinery. To direct expression during baculovirus infection, the above promoter/enhancer combination is active during the early stage of baculovirus infection, and an additional p10 promoter directs expression in the late/very late phases. We also created additional vectors featuring alternative combinations of baculovirus-derived promoters and enhancers to verify that our particular combination of enhancer and promoter elements was optimal. Using the Radiance Ek/LIC cloning method, the reporter enzyme Renilla luciferase (Rluc) was cloned into all variants to allow comparisons of relative expression strength. The plasmid that gave the best overall performance in both systems was chosen as our new dual-purpose expression plasmid, pIEx/Bac. To further test the vector, we cloned additional inserts encoding differing classes of proteins, including an importin, a phosphatase, and kinases. The results demonstrate the utility of the pIEx/Bac vector for streamlined expression in insect cells.

A fundamental challenge in high-throughput (HT) expression screening is to rapidly identify the appropriate expression system for many targets in parallel. Known or unknown open reading frames (ORFs) are typically amplified by PCR and then cloned into a variety of vectors, producing recombinants used to direct target protein expression in Escherichia coli , insect cells, mammalian cells, or yeast. To facilitate rapid expression and purification in Spodoptera insect cells ( Sf 9), we developed transient expression vectors that include an enterokinase cleavage site immediately upstream of a ligation-independent cloning site (Ek/LIC). We also developed a high-efficiency insect cell transfection reagent, and automation-compatible fusion protein purification system for insect cells to facilitate expression screening and protein production. Positive clones identified from the small-scale screening were subjected to a larger scale production. Using this InsectDirect TM approach, we successfully expressed milligram quantities of different human proteins including heat shock proteins, phospholipases, and protein kinases.