Today, many thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Yet, in addition to its value in producing recombinant proteins in insect cells and larvae, this viral vector system continues to evolve in new and unexpected ways. This is exemplified by the development of engineered insect cell lines to mimic mammalian cell glycosylation of expressed proteins, baculovirus display strategies and the application of the virus as a mammalian-cell gene delivery vector. Novel vector design and cell engineering approaches will serve to further enhance the value of baculovirus technology.

/pdf/baculovirus-as-versatile-vectors-for-protein-expression-in-1x56ppbq8n.pdf

Baculovirus as versatile vectors for protein expression in insect and mammalian cells

https://geneva-biotech.com/wp-content/uploads/2016/04/Simon-Trowitzsch-2010.-J.-Structural-Biology.pdf

New baculovirus expression tools for recombinant protein complex production.

From gene to protein: a review of new and enabling technologies for multi-parallel protein expression

Recent advances in the production of proteins in insect and mammalian cells for structural biology

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.

/pdf/eukaryotic-expression-developments-for-structural-proteomics-1o8dyczgwc.pdf

Eukaryotic expression: developments for structural proteomics.

Optimisation of insect cell growth in deep-well blocks: development of a high-throughput insect cell expression screen

The Baculovirus Expression Vector System (BEVS) is one of the most efficient systems for production of recombinant proteins and consequently its application is wide-spread in industry as well as in academia Since the early 1970s, when the first stable insect cell lines were established and the infectivity of bacu-lovirus in an in vitro culture system was demonstrated (1, 2), virtually thousands of reports have been published on the successful expression of proteins using this system as well as on method improvement However, despite its popularity the system is labor intensive and time consuming Moreover, adaptation of the system to multi-parallel (high-throughput) expression is much more difficult to achieve than with E coli due to its far more complex nature However, recent years have seen the development of strategies that have greatly enhanced the stream-lining and speed of baculovirus protein expression for increased throughput via use of automation and miniaturization This chapter therefore tries to collate these developments in a series of protocols (which are modifications to standard procedure plus several new approaches) that will allow the user to expedite the speed and throughput of baculovirus-mediated protein expression and facilitate true multi-parallel, high-throughput protein expression profiling in insect cells In addition we also provide a series of optimized protocols for small and large-scale transient insect cell expression that allow for both the rapid analysis of multiple constructs and the concomitant scale-up of those selected for on-going analysis Since this approach is independent of viral propagation, the timelines for this approach are markedly shorter and offer a significant advantage over standard bacu-lovirus expression approach strategies in the context of HT applications

High-throughput insect cell protein expression applications.

The use of Chinese Hamster Ovary (CHO) cells for transient gene expression is gaining importance steadily, as it has been shown that both quality and quantity of proteins derived from CHO cells differs from and can even be superior to material derived from Human Embryonic Kidney (HEK293) cells [1-4]. In order to augment yields HEK cell lines have been genetically modified, by e.g. stable integration of an expression cassette coding for the Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) in conjunction with expression plasmid vectors containing the EBNA1 interaction site oriP. Here we describe the generation of a CHO cell line stably expressing the EBNA1 gene to enhance yields after large scale transient transfection with polyethylenimine (PEI).

An expression plasmid featuring the EBNA1 gene was transfected into an in-house available CHO wild type cell line by nucleofection and several stable pools were established by antibiotic selection. In a first approach, 133 clones were then isolated by limiting dilution cloning and subsequently expanded for further analysis. The approach aimed at identifying clones showing both the presence of EBNA1 protein and enhanced yields after PEI-mediated transient expression of a reporter protein at the same time.

Cell lysates or nuclear and cytoplasmic protein extracts from these clones were tested for presence of EBNA1 protein by Western Blot. An in-licensed cell line, CHO EBNALT85 (Icosagen AS, Tartu, Estonia) expressing the full length EBNA1 gene, and the HEK293-6E cell line (from the group of Y. Durocher, NRC, Canada) expressing a truncated EBNA1 gene served as positive controls. A number of commercially available anti-EBNA1 antibodies were tested in Western blotting, but most antibodies failed to detect the EBNA1 protein produced by these positive control cells. Only one antibody (Antibody 1EB12, Santa Cruz Biotechnology) was identified that detected the EBNA1 proteins in both positive control cell lines.

However, by applying this antibody on the 133 generated clones it became obvious that in most of them the EBNA1 protein appeared to be largely degraded.

The functionality of the EBNA1 protein in the cells was probed by PEI-mediated transient transfection of expression plasmids encoding the gene for the reporter protein Secreted Alkaline Phosphatase (SeAP). SeAP expression levels were compared with respect to presence or absence of the EBNA1 interaction sequence oriP on the SeAP expression plasmid. Only one clone showed a twofold enhanced production level of the reporter protein compared to the non-engineered parental CHO cell line. However, in this specific clone no EBNA1 protein could be detected by Western Blot, and the enhanced expression level was independent from the presence or absence of the oriP sequence on the SeAP expression plasmid. We therefore conclude that this clone does not possess a functional EBNA1 protein and an EBNA1/oriP based enhancement of productivity. The mechanism underlying the enhanced yield of the reporter protein in this particular clone remains currently elusive.

In an alternative approach flow cytometric cell sorting was applied to generate CHO clones producing a functional EBNA1 protein. Using the EBNA1 positive CHO cell line CHO EBNALT85 it could be shown that expressing eGFP from a transiently transfected expression plasmid with oriP sequence resulted in a 30- to 40-fold increase of highly fluorescent cells in comparison to an expression plasmid lacking the oriP sequence.

Thus, the already generated CHO pools stably transfected with the EBNA1 gene were transiently supertransfected with an eGFP expression plasmid containing the oriP sequence. Highly fluorescent cells were collected by cell sorting and expanded. Analysis by Western Blot revealed that this sub-pool was enriched for cells exhibiting the presence of a full length EBNA1 protein besides a major breakdown product. Transient transfection of this sub-pool with SeAP expression vectors led to the conclusion that the pool contained cells with a functional EBNA1 protein capable of enhancing SeAP yields in dependence on the presence of the oriP sequence on the SeAP expression plasmid. Clones were again generated from this sub-pool by limiting dilution cloning and characterized in analogy to the first approach. 9 out of 12 clones that were analyzed further showed increased levels of reporter protein production in dependence on the presence of the oriP element on the expression plasmid. The series of clones exhibiting highest expression levels will now be further evaluated by expression of other protein candidates.

https://link.springer.com/content/pdf/10.1186%2F1753-6561-5-S8-P132.pdf

About making a CHO production cell line “research-friendly” by genetic engineering

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Optimisation of insect cell growth in deep-well blocks: development of a high-throughput insect cell expression screen

High-throughput insect cell protein expression applications.

About making a CHO production cell line “research-friendly” by genetic engineering