Scarless Baculovirus Genome Editing Using Lambda-Red Recombineering in E. coli.

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.

Re-Analysis of the Widely Used Recombinant Murine Cytomegalovirus MCMV-m157luc Derived from the Bacmid pSM3fr Confirms Its Hybrid Nature.

Murine cytomegalovirus (MCMV), and, in particular, recombinant virus derived from MCMV-bacmid pSM3fr, is widely used as the small animal infection model for human cytomegalovirus (HCMV). We sequenced the complete genomes of MCMV strains and recombinants for quality control. However, we noticed deviances from the deposited reference sequences of MCMV-bacmid pSM3fr. This prompted us to re-analyze pSM3fr and reannotate the reference sequence, as well as that for the commonly used MCMV-m157luc reporter virus. A correct reference sequence for this frequently used pSM3fr, containing a repaired version of m129 (MCK-2) and the luciferase gene instead of ORF m157, was constructed. The new reference also contains the original bacmid sequence, and it has a hybrid origin from MCMV strains Smith and K181.

Recombinant Human Cytomegalovirus Expressing an Analog-Sensitive Kinase pUL97 as Novel Tool for Functional Analyses.

The human cytomegalovirus (HCMV) is a member of the beta-herpesvirus family and inflicts life-long latent infections in its hosts. HCMV has been shown to manipulate and dysregulate many cellular processes. One major interactor with the cellular host is the viral kinase pUL97. The UL97 gene is essential for viral replication, and kinase-deficient mutants of pUL97 display a severe replication defect. Recently, another group established an analog-sensitive version of the pUL97 protein. This mutant kinase can be treated with a non-hydrolysable ATP analog, thereby inhibiting its kinase function. This process is reversible by removing the ATP analog by media change. We introduced this mutant version of the pUL97 protein into the laboratory strain Ad169 of HCMV, BADwt, creating a BAD-UL97-as1 viral mutant. This mutant virus replicated normally in infected cells in the absence of the ATP analog and maintained its ability to phosphorylate its cellular substrates. However, when treated with the ATP analog, BAD-UL97-as1 displayed a defect in the production of intra- and extracellular viral DNA and in the production of viral progeny. Furthermore, in the presence of 3MB-PP1, a well-established substrate of pUL97 was no longer hyperphosphorylated. This effect was detectable as early as 4 h post treatment, which allows for studies on pUL97 without the complication of low viral titers. Nevertheless, we observed off-target effects of 3MB-PP1 on several cellular processes, which should be considered with this approach.

Rescue of Recombinant Adenoviruses by CRISPR/Cas-Mediated in vivo Terminal Resolution.

Recombinant adenovirus (rAd) vectors represent one of the most frequently used vehicles for gene transfer applications in vitro and in vivo. rAd genomes are constructed in Escherichia coli where their genomes can be maintained, propagated, and modified in form of circular plasmids or bacterial artificial chromosomes. Although the rescue of rAds from their circular plasmid or bacmid forms is well established, it works with relatively low primary efficiency, preventing this technology for library applications. To overcome this barrier, we tested a novel strategy for the reconstitution of rAds that utilizes the CRISPR/Cas-machinery to cleave the circular rAd genomes in close proximity to their inverted terminal repeats (ITRs) within the producer cells upon transfection. This CRISPR/Cas-mediated in vivo terminal resolution allowed efficient rescue of vectors derived from different human adenovirus (HAdV) species. By this means, it was not only possible to increase the efficiency of virus rescue by about 50-fold, but the presented methodology appeared also remarkably simpler and faster than traditional rAd reconstitution methods.

Purification and Characterization of Human DNA Ligase IIIα Complexes After Expression in Insect Cells.

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.

A review of alternative promoters for optimal recombinant protein expression in baculovirus-infected insect cells.

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.

Relocation of the attTn7 Transgene Insertion Site in Bacmid DNA Enhances Baculovirus Genome Stability and Recombinant Protein Expression 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.

Construction of the Antheraea pernyi (Lepidoptera: Saturniidae) Multicapsid Nucleopolyhedrovirus Bacmid System.

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.

Protein Expression in the Baculovirus-Insect Cell Expression System.

The baculovirus-insect cell expression system is a popular choice for recombinant protein production. Post-translational modifications, production of protein complexes, and reported high protein yields are some of the favorable features of this eukaryotic expression system. The intricacies of the baculovirus-insect cell expression system may deter beginners from implementing it for routine protein production. This chapter documents a detailed protocol for using the baculovirus-insect cell expression system, which has been verified in a protein production research facility.

Neospora caninum antigens displaying virus-like particles as a bivalent vaccine candidate against neosporosis.

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.

Development of SpyTag/SpyCatcher-Bacmid Expression Vector System (SpyBEVS) for Protein Bioconjugations Inside of Silkworms.

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.

Formation of Virus-Like Particles of the Dengue Virus Serotype 2 Expressed in Silkworm Larvae.

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.

Secretory Nanoparticles of Neospora caninum Profilin-Fused with the Transmembrane Domain of GP64 from Silkworm Hemolymph.

Neosporosis, which is caused by Neospora caninum, is a well-known disease in the veterinary field. Infections in pregnant cattle lead to abortion via transplacental (congenitally from mother to fetus) transmission. In this study, a N. caninum profilin (NcPROF), was expressed in silkworm larvae by recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid and was purified from the hemolymph. Three NcPROF constructs were investigated, native NcPROF fused with an N-terminal PA tag (PA-NcPROF), PA-NcPROF fused with the signal sequence of bombyxin from B. mori (bx-PA-NcPROF), and bx-PA-NcPROF with additional C-terminal transmembrane and cytoplasmic domains of GP64 from BmNPV (bx-PA-NcPROF-GP64TM). All recombinant proteins were observed extra- and intracellularly in cultured Bm5 cells and silkworm larvae. The bx-PA-NcPROF-GP64TM was partly abnormally secreted, even though it has the transmembrane domain, and only it was pelleted by ultracentrifugation, but PA-NcPROF and bx-PA-NcPROF were not. Additionally, bx-PA-NcPROF-GP64TM was successfully purified from silkworm hemolymph by anti-PA agarose beads while PA-NcPROF and bx-PA-NcPROF were not. The purified bx-PA-NcPROF-GP64TM protein bound to its receptor, mouse Toll-like receptor 11 (TLR-11), and formed unique nanoparticles. These results suggest that profilin fused with GP64TM was secreted as a nanoparticle with binding affinity to its receptor and this nanoparticle formation is advantageous for the development of vaccines to N. caninum.

Purification of human papillomavirus-like particles expressed in silkworm using a Bombyx mori nucleopolyhedrovirus bacmid expression system.

A three-stage chromatography protocol for the purification of human papillomavirus-like particles (HPV-LPs) from the silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system was developed. For host cell DNA separation, anion exchange chromatography was used after screening for a suitable stationary phase. Using the two separation principles of cation exchange chromatography and metal affinity of ceramic hydroxyapatite (CHT) as a second stage, the amount of baculovirus in the sample was reduced to less than the detection limit of qPCR. The CHT separation was optimized with respect to the elution buffer used; 150-600 mM sodium phosphate, pH 7.2, resulted in the highest recovery of HPV-LPs. Using heparin chromatography, it was possible to reduce the sample volume and to thus highly concentrate the target protein during the separation of contaminating proteins. During the second purification stage, over 99.3% of the DNA was removed, and no infectious baculoviruses remained. After concentration by heparin column chromatography, over 99.9% of the DNA and protein had been removed. The purity achieved by this method exceeds that obtained by DDDDK-tag-based affinity chromatography and sucrose gradient ultracentrifugation, which were used as comparative purification methods. The 3-stage purification of HPV-LPs from silkworm fat bodies described here was a proof of concept and is a scalable method, but the overall yield remains to be improved.

Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Heterologous expression, purification and characterization of human ß-1,2-N-acetylglucosaminyltransferase II using a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system.

ß-1,2-N-Acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi-localized type II transmembrane enzyme that catalyzes the transfer of N-acetylglucosamine to the 6-arm of the trimanosyl core of N-glycans, an essential step in the conversion of oligomannose-type to complex-type N-glycans. Despite its physiological importance, there have been only a few reports on the heterologous expression and structure-function relationship of this enzyme. Here, we constructed a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system and expressed human GnTII (hGnTII) lacking the N-terminal cytosolic tail and transmembrane region. The recombinant hGnTII was purified from silkworm larval hemolymph in two steps by using tandem affinity purification tags, with a yield of approximately 120 µg from 10 mL hemolymph, and exhibited glycosyltransferase activity and strict substrate specificity. The enzyme was found to be N-glycosylated by the enzymatic cleavage of glycans, while hGnTII expressed in insect cells had not been reported to be glycosylated. Although insects typically produce pauci-mannosidic-type glycans, the structure of N-glycans in the recombinant hGnTII was suggested to be of the complex type, and the removal of the glycans did not affect the enzymatic activity.

Designing of a Recombinant Agip Bacmid Construct with Infectious Properties Against Black Cutworm Agrotis ipsilon Larvae.

In this study, Agrotis ipsilon nucleopolyhedrovirus bacmid has been constructed as an infectious bacmid in an attempt to allow genome recombination and generation of virus mutants. Since the FseI, a unique restriction site, is located in a viral coding region (ORF_119), PCR was performed to partially amplify the ORF_119 fragment containing the FseI site to facilitate the bacmid construction in a proper way without interrupting the ORF expression. Construction with repeated fragments at the end of the cloned viral was carried out in an attempt to facilitate circulation during infection in insect cells. The amplified gp_119 fragment was cloned into the BAC_Bsu361 plasmid derived from the AcMNPV Bac-to-Bac® system. Recombinant plasmid was used to subclone the Agrotis ipsilon nucleopolyhedrovirus (AgipNPV)-linearized genome using the FseI unique site. The Agip bacmid DNA extracted from Escherichia coli was used to transfect A. ipsilon third instar larvae by injection into the hemolymph. The produced occlusion bodies were purified from infected larvae and used to feed healthy larvae for amplifying the virus, and infectivity was recorded. Using bacmid technology will facilitate manipulation of the AgipNPV genome and help in determining the genetic factors involved in virus virulence and biology.

Introduction of cationic virosome derived from vesicular stomatitis virus as a novel gene delivery system for sf9 cells.

Insect-derived cell lines are used extensively to produce recombinant proteins because they are capable of performing a range of post-translational modifications. Due to their significance in biotechnological applications, various methods have been developed to transfect them. In this study, we introduce a virosome constructed from vesicular stomatitis virus (VSV) as a new delivery system for sf9 cells. We labeled these VSV virosomes by fluorescent probe Rhodamine B chloride (R18). By fluorescence microscope observation and conducting a fusion assay, we confirmed the uptake of VSV virosomes via endocytosis by sf9 cells and their fusion with the endosomal membrane. Moreover, we incubated cationic VSV virosomes with a GFP-expressing bacmid and transfected sf9 cells, after 24 h some cells expressed GFP indicating the ability of VSV virosomes to deliver heterologous DNA to these cells. This is the first report of a virosome-based delivery system introduced for an insect cell line.

Designing Two Individual AcMNPV Polyhedrin-Plus Bac-to-Bac Expression System in order to Express GFP and CPV-VP2 in Insect Cells.

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.

Recombinant expression and purification of functional vascular endothelial growth factor-121 in the baculovirus expression system.

OBJECTIVE: To express human vascular endothelial growth factor121 (VEGF121) in insect cells. METHODS: A gene construct containing VEGF was cloned in the pFastBac-HTA vector, followed by transformation in DH10BAC. The recombinant bacmid was then extracted, and transfected into Sf9 insect cells. The transfected cells were harvested, and then VEGF expression was confirmed by western blotting using specific antibodies. The tube formation assay was used for functional assessment of VEGF. RESULTS: Our results showed that VEGF could be successfully expressed in the baculovirus system. Purified VEGF was able to stimulate in vitro tube formation of human endothelial cells. CONCLUSIONS: Results from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes.