Baculoviruses are widely used for their potential as biological pesticide and as platform for the production of recombinant proteins and gene therapy vectors. The Baculovirus Expression Vector System (BEVS) is used for high level of expression of (multiple) proteins in insect cells. Baculovirus recombinants can be quickly constructed by transposition of the gene(s) of interest into a so-called bacmid, which is a baculovirus infectious clone maintained as single-copy, bacterial artificial chromosome in Escherichia coli. A two-step homologous recombineering technique using the lambda-red system in E. coli allows for scarless editing of the bacmid with PCR products based on sequence homology. In the first step, a selection cassette with 50 bp homology arms, typically generated by PCR, is inserted into the designated locus. In the second step, the selection cassette is removed based on a negative selection marker, such as SacB or rpsL. This lambda-red recombineering technique can be used for multiple gene editing purposes, including (large) deletions, insertions, and even single point mutations. Moreover, since there are no remnants of the editing process, successive modifications of the same bacmid are possible. This chapter provides detailed instructions to design and perform two-step homologous recombineering of baculovirus bacmid DNA in E. coli. We present two case studies demonstrating the utility of this technique for creating a deletion mutant of the chitinase and cathepsin genes and for introducing a single point mutation in the baculovirus gene gp41. This scarless genome editing approach can facilitate functional studies of baculovirus genes and improve the production of recombinant proteins using the BEVS.

With improvements in biophysical approaches, there is growing interest in characterizing large, flexible multi-protein complexes. The use of recombinant baculoviruses to express heterologous genes in cultured insect cells has advantages for the expression of human protein complexes because of the ease of co-expressing multiple proteins in insect cells and the presence of a conserved post-translational machinery that introduces many of the same modifications found in human cells. Here we describe the preparation of recombinant baculoviruses expressing DNA ligase IIIα, XRCC1, and TDP1, their subsequent co-expression in cultured insect cells, the purification of complexes containing DNA ligase IIIα from insect cell lysates, and their characterization by multi-angle light scattering linked to size exclusion chromatography and negative stain electron microscopy.

Generating recombinant proteins in insect cells has been made possible via the use of the Baculovirus Expression Vector System (BEVS). Despite the success of many proteins via this platform, some targets remain a challenge due to issues such as cytopathic effects, the unpredictable nature of co-infection and co-expressions, and baculovirus genome instability. Many promoters have been assayed for the purpose of expressing diverse proteins in insect cells, and yet there remains a lack of implementation of those results when reviewing the landscape of commercially available baculovirus vectors. In advancing the platform to produce a greater variety of proteins and complexes, the development of such constructs cannot be avoided. A better understanding of viral gene regulation and promoter options including viral, synthetic, and insect-derived promoters will be beneficial to researchers looking to utilize BEVS by recruiting these intricate mechanisms of gene regulation for heterologous gene expression. Here we summarize some of the developments that could be utilized to improve the expression of recombinant proteins and multi-protein complexes in insect cells.

Baculovirus expression vectors are successfully used for the commercial production of complex (glyco)proteins in eukaryotic cells. The genome engineering of single-copy baculovirus infectious clones (bacmids) in E. coli has been valuable in the study of baculovirus biology, but bacmids are not yet widely applied as expression vectors. An important limitation of first-generation bacmids for large-scale protein production is the rapid loss of gene of interest (GOI) expression. The instability is caused by the mini-F replicon in the bacmid backbone, which is non-essential for baculovirus replication in insect cells, and carries the adjacent GOI in between attTn7 transposition sites. In this paper, we test the hypothesis that relocation of the attTn7 transgene insertion site away from the mini-F replicon prevents deletion of the GOI, thereby resulting in higher and prolonged recombinant protein expression levels. We applied lambda red genome engineering combined with SacB counterselection to generate a series of bacmids with relocated attTn7 sites and tested their performance by comparing the relative expression levels of different GOIs. We conclude that GOI expression from the odv-e56 (pif-5) locus results in higher overall expression levels and is more stable over serial passages compared to the original bacmid. Finally, we evaluated this improved next-generation bacmid during a bioreactor scale-up of Sf9 insect cells in suspension to produce enveloped chikungunya virus-like particles as a model vaccine.

In this study, we established the Antheraea pernyi multicapsid nucleopolyhedrovirus (AnpeNPV) bacmid system for the construction of a Bac-to-Bac expression system and the generation of virus mutants. The CopyRight pSMART BAC cloning vector harboring the chloramphenicol resistance gene was introduced into the AnpeNPV genome to produce the AnpeNPV bacmid that could be propagated in Escherichia coli with stable replication. The enhanced green fluorescent protein (EGFP) was successfully expressed in both Tn-Hi5 cells and A. pernyi pupae using the AnpeNPV Bac-to-Bac expression system. To generate the AnpeNPV mutants, we developed the AnpeNPV bacmid/λ Red recombination system that facilitated the deletion of viral genes from the AnpeNPV genome. The genes cathepsin and chitinase were deleted and a derivative AnpeNPV Bac-to-Bac expression system was constructed. Furthermore, we demonstrated that the novel expression system could be used to express human epidermal growth factor in A. pernyi pupae. Taken together, the AnpeNPV bacmid system provides a powerful tool to create the AnpeNPV Bac-to-Bac expression system for protein expression in A. pernyi pupae. Further, it helps to knock-out genes from the AnpeNPV genome with λ Red recombination system for identification of the role of viral genes involved in regulating gene expression, DNA replication, virion structure, and infectivity during the AnpeNPV infection process.

Neospora caninum is a causative and transmissible agent of dog and bovine neosporosis. The resulting reproductive failures in infected cattle lead to significant economic losses worldwide. However, there is no satisfactory treatment or vaccine currently available to combat this pathogen. Thus, the development of appropriate vaccines to manage its infection and transmission is urgently needed. In this study, we expressed Rous sarcoma virus-like particles (RSV-LP) that displayed dual N. caninum antigens in silkworms. The antigen candidates are modified by adding a transmembrane domain of GP64 protein from Bombyx mori nucleopolyhedrovirus (BmNPV) to the C-terminus of surface antigen 1 (NcSAG1) and SAG1-related sequence 2 (NcSRS2). The NcSRS2 alone or the NcSAG1/NcSRS2 bivalent form displaying RSV-LPs were purified using sucrose density gradient centrifugation. These purified VLPs were then used for immunizations in gerbils, Meriones unguiculatus, to evaluate the anti-N. caninum effects in vivo. The results demonstrated that antigens displaying RSV-LPs in immunized gerbils produced the antigen-specific antibody, leading to a relatively lower parasite load after infections of N. caninum. To the best of our knowledge, this is the first study to present an RSV-LP vaccine displaying bivalent antigens from neosporosis. Taken together, our strategy suggests that silkworm-expressed virus-like particles (VLPs) are promising bivalent vaccine candidates against N. caninum infections.

To explore virus-like particles formation of dengue virus serotype type 2 (DENV-2) structural proteins of, C, prM, E were expressed in silkworm larvae using recombinant Bombyx mori nucleopolyhedroviruses (BmNPV). Each recombinant BmNPV bacmid coding the 2C-prM-E polypeptide and E protein fused with the signal peptide of bombyxin from B. mori was injected into silkworm larvae. The expressed proteins were collected from hemolymph and fat body, and purified using affinity chromatography. E protein was observed at 55 kDa. The DENV virus-like particles (DENV-LPs) with a diameter approximately 35 nm was observed using transmission electron microscopy (TEM) and immunogold-labelling TEM analysis. The binding of each partially purified proteins to heparin, one of receptors for DENV was confirmed. DENV-LPs were secreted in silkworm larval hemolymph even still low amount, but the E protein and heparin binding function were confirmed.

Protein conjugations at post-translational levels are known to be essential to protein stability and function. Recently, it has been proven that the split protein CnaB2 (SpyTag/SpyCatcher, ST/SC) from Streptococcus pyogenes can induce covalent conjugation rapidly and efficiently under various conditions. The protein of interest fused with the split protein SC/ST could be assembled spontaneously. In light of this finding, we introduced the ST/SC protein coupling concept into the silkworm-bacmid protein expression system (SpyBEVS). As a proof of concept, we first examined and confirmed that a competent ligation occurred between ST/SC-fused protein partners in vitro in cultured silkworm cells and in vivo in silkworm larvae by co-infection of several recombinant baculoviruses. The protein conjugation could be also achieved sufficiently by a simple one-step mixture of purified ST/SC-tagged peptide-protein pairs in vitro. Given the flexibility and robustness of silkworm-BEVS, our results on SpyBEVS show an alternative method for enabling the production of protein decorations in vitro and inside of silkworms.

Background: The importance of viral protein-2 (VP2) of canine parvovirus (CPV) in binding to human cancer cells, production of veterinary vaccines and diagnostic kits has motivated several researches on producing this protein. Objectives: Our purpose was to construct recombinant bacmid shuttle vectors expressing VP2 of CPV using Bac-to-Bac baculoviral expression system. Materials and Methods: Mini-Tn7 transposones engineered in pFastBac1 donor vectors were used to construct expression cassettes of GFP and CPV-VP2. The plasmids were transferred into E. coli DH10Bac competent cells. Site-specific transposition of the genes into bacmid was accomplished using helper plasmid. Occurrence of Transposition was confirmed via PCR using specific primers and PUC/M13 universal primers. The recombinant bacmid DNAs were transfected into Sf9 cells using cationic lipids to generate new recombinant baculoviruses expressing GFP and CPV-VP2. GFP and VP2 expressions were evaluated by fluorescence microscopy and western analysis, respectively. Results: Cloning, subcloning and recombination processes of both GFP and VP2 were accomplished and verified. Accuracy of transfection process was confirmed by GFP fluorescence microscopy.VP2 expression was verified by SDS-PAGE and western analysis. Conclusions: Two Bac-to-Bac expression systems were designed to produce recombinant VP2 and GFP in insect cells.

The Baculovirus expression vector system (BEVS) is a transient expression platform for recombinant protein production in insect cells. Baculovirus infection of insect cells will shutoff host translation and induce apoptosis and lead to the termination of protein expression. Previous reports have demonstrated the enhancement of protein yield in BEVS using stable insect cell lines expressing interference RNA to suppress the expression of caspase-1.
In this study, short-hairpin RNA (shRNA) expression cassettes targeting Spodoptera frugiperda caspase-1 (Sf-caspase-1) were constructed and inserted into an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vector. Using the recombinant baculovirus vectors, we detected the suppression of Sf-caspase-1 expression and cell apoptosis. Green fluorescent protein (GFP), Discosoma sp. Red (DsRed) and firefly luciferase were then expressed as reporter proteins. The results showed that suppression of apoptosis enhanced the accumulation of exogenous proteins at 2 and 3 days post infection. After 4 days post infection, the activity of the reporter proteins remained higher in BEVS using the baculovirus carrying shRNA in comparison with the control without shRNA, but the accumulated protein levels showed no obvious difference between them, suggesting that apoptosis suppression resulted in improved protein folding rather than translation efficiency at the very late stage of baculovirus infection.
The baculovirus vector developed in this study would be a useful tool for the production of active proteins suitable for structural and functional studies or pharmaceutical applications in Sf9 cells, and it also has the potential to be adapted for the improvement of protein expression in different insect cell lines that can be infected by AcMNPV.