Mammalian cell lines are one of the best options when it comes to the production of complex proteins requiring specific glycosylation patterns. Plasmid DNA transfection and stable cell lines are frequently used for recombinant protein production, but they are expensive at large scale or can become time-consuming, respectively. The BacMam baculovirus (BV) is a safe and cost-effective platform to produce recombinant proteins in mammalian cells. The process of generating BacMam BVs is straightforward and similar to the generation of \"insect\" BVs, with different commercially available platforms. Although there are several protocols that describe recombinant protein expression with the BacMam BV in adherent cell lines, limited information is available on suspension cells. Therefore, it is of relevance to define the conditions to produce recombinant proteins in suspension cell cultures with BacMam BVs that facilitate bioprocess transfer to larger volumes. Here, we describe a method to generate a high titer BacMam BV stock and produce recombinant proteins in suspension HEK293 cells.

Efficient genome editing by using CRISPR technologies requires simultaneous and efficient delivery of multiple genetically encoded components to mammalian cells. Amongst all editing approaches, prime editing (PE) has the unique potential to perform seamless genome rewriting, in the absence of DNA double-strand breaks (DSBs). The cargo capacity required for efficient PE delivery to mammalian cells stands at odd with the limited packaging capacity of traditional viral delivery vectors. By contrast, baculovirus (BV) has a large synthetic DNA capacity and can efficiently transduce mammalian cells. Here we describe a protocol for the assembly of baculovirus vectors for multiplexed prime editing in mammalian cells.

This chapter outlines a rapid detection method to determine the virus titer of your baculovirus stock. Staining of cells with fluorescently labeled gp64 antibody allows for flow cytometer-based quantitation of baculovirus-infected insect cells. In this assay, Sf9 cells are infected with tenfold serial dilutions of the test virus stock, and baculovirus titers are calculated based on the ratio of infected to uninfected cells 13 to 18 h after inoculation.

There are many methods that can be used to determine the infectious titer of your baculovirus stock. The TCID50 method is a simple end-point dilution method that determines the amount of baculovirus virus needed to produce a cytopathic effect or kill 50% of inoculated insect cells. Serial dilutions of baculovirus stock are added to Sf9 cells cultivated in 96-well plates and 3-5 days after infection, cells are monitored for cell death or cytopathic effect. The titer can then be calculated by the Reed-Muench method as described in this method.

Plaque assay method enables the quantification of infectious baculovirus when defined as plaque forming units (PFU). It allows to determine the amount of infectious virus needed to infect the cells at a specific multiplicity of infection (MOI). Serial dilutions of baculovirus stock are added to the Sf9 cells monolayer followed by addition of 5% Agarose overlay. Six days after infection clear infection halos are observed using a neutral red solution. Here we describe the quantification of recombinant baculovirus expression vector (rBEV) carrying a transgene in an rAAV expression cassette. Reproducible quantification of PFU is obtained with this method.

The Baculovirus Expression Vector System (BEVS) is used with cultured insect cells to produce a wide variety of heterologous proteins, which can be secreted into the culture medium during the transient infection process (Smith et al. Mol Cell Biol 12:2156-2165, 1983). When the infection process is complete, centrifugation is often used to separate the desired protein from the spent insect cells. The desired product in the harvested supernatant is contaminated with baculovirus, amino acids, lipids, detergents, oils, lysed cells from the infection process, genomic DNA from the insect cells, and proteases due to the lytic nature of the baculovirus infection process and many other contaminants (Ikonomou et al. Appl Microbiol Biotechnol 62:1-20, 2003). All these contaminants that are present in the centrifuged supernatant with the desired secreted protein make the initial chromatographic capture step critical for effective purification of the desired protein. A purification scheme will be outlined for a slightly acidic secreted protein using cation exchange chromatography (Lundanes et al. Chromatography: basic principles, sample preparations and related methods, 1st edn. Wiley, 2013).

The insect cell-baculovirus expression vector (IC-BEV) platform has enabled small research-scale and large commercial-scale production of recombinant proteins and therapeutic biologics including recombinant adeno-associated virus (rAAV)-based gene delivery vectors. The wide use of this platform is comparable with other mammalian cell line-based platforms due to its simplicity, high-yield, comparable quality attributes, and robust bioprocessing features. In this chapter, we describe a rAAV production protocol employing one of the recent modifications of the One-Bac platform that consists of a stable transformed Sf9 cell line carrying AAV Rep2/Cap5 genes that are induced upon infection with a single recombinant baculovirus expression vector harboring the transgene of interest (rAAV genome). The overall protocol consists of essential steps including rBEV working stock preparation, rAAV production, and centrifugation-based clarification of cell culture lysate. The same protocol can also be applied for rAAV vector production using traditional Three-Bac, Two-Bac, and Mono-Bac platforms without requiring significant changes.

Insect cell expression has been successfully used for the production of viral antigens as part of commercial vaccine development. As expression host, insect cells offer advantage over bacterial system by presenting the ability of performing post-translational modifications (PTMs) such as glycosylation and phosphorylation thus preserving the native functionality of the proteins especially for viral antigens. Insect cells have limitation in exactly mimicking some proteins which require complex glycosylation pattern. The recent advancement in insect cell engineering strategies could overcome this limitation to some extent. Moreover, cost efficiency, timelines, safety, and process adoptability make insect cells a preferred platform for production of subunit antigens for human and animal vaccines. In this chapter, we describe the method for producing the SARS-CoV2 spike ectodomain subunit antigen for human vaccine development and the virus like particle (VLP), based on capsid protein of porcine circovirus virus 2 (PCV2d) antigen for animal vaccine development using two different insect cell lines, SF9 & Hi5, respectively. This methodology demonstrates the flexibility and broad applicability of insect cell as expression host.

The baculovirus expression vector system (BEVS) is a powerful platform for protein expression in insect cells. A prevalent application is the expression of complex protein structures consisting of multiple, interacting proteins. Coinfection with multiple baculoviruses allows for production of complex structures, facilitating structure-function studies, allowing augmentation of insect cell functionality, and production of clinically relevant products such as virus-like particles (VLPs) and adeno-associated viral vectors (AAV). Successful coinfections require the generation of robust and well-quantified recombinant baculovirus stocks. Virus production through homologous recombination, combined with rigorous quantification of viral titers, allows for synchronous coinfections producing high end-product titers. In this chapter, we describe the streamlined workflow for generation and quantification of high-quality recombinant baculovirus stocks and successful coinfection as defined by a preponderance of dually infected cells in the insect cell culture.

The baculovirus expression vector system (BEVS) has now found acceptance in both research laboratories and industry, which can be attributed to many of its key features including the limited host range of the vectors, their non-pathogenicity to humans, and the mammalian-like post-translational modification (PTMs) that can be achieved in insect cells. In fact, this system acts as a middle ground between prokaryotes and higher eukaryotes to produce complex biologics. Still, industrial use of the BEVS lags compared to other platforms. We have postulated that one reason for this has been a lack of genetic tools that can complement the study of baculovirus vectors, while a second reason is the co-production of the baculovirus vector with the desired product. While some genetic enhancements have been made to improve the BEVS as a production platform, the genome remains under-scrutinized. This chapter outlines the methodology for a CRISPR-Cas9-based transfection-infection assay to probe the baculovirus genome for essential/nonessential genes that can potentially maximize foreign gene expression under a promoter of choice.