Abstract
Baculovirus expression vector systems (BEVS) are widely used to produce heterologous proteins for a wide range of applications. Developed more than 30 years ago, BEVS have been constantly modified to improve product quality and ease-of-use. Plasmid reagents were tailored and engineered to facilitate introduction of heterologous genes into baculoviral genomes. At the same time, detrimental modalities such as genes encoding proteases or apoptotic factors were removed to improve protein yield. Advances in DNA synthesis and manipulation now enable the engineering of part or whole synthetic baculovirus genomes, opening up new avenues to redesign and tailor the system to specific applications. Here, we describe a simple protocol for designing and constructing baculovirus genomes comprising segments of synthetic DNA through the use of iterative Red/ET homologous recombination reactions.
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Acknowledgments
We thank all members of the Berger laboratory for their contributions. We are grateful to Robert Roth (AstraZeneca) for helpful discussions. The authors thank Francis Stewart for the pRed/ET plasmid. B.G. is supported by the Biotechnology and Biological Research Council (BBSRC) through a scholarship from the South West Doctoral Training Programme, SWDTP. I.B. is supported by a European Research Council ERC Advanced Grant (DNA-DOCK) and is recipient of a Wellcome Trust Senior Investigator Award.
Competing Financial Interest Statement
The authors declare competing financial interest. I.B. is inventor on patents protecting MultiBac. I.B. is also shareholder of biotech companies commercializing MultiBac applications.
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Crocker, H., Gorda, B., Pelosse, M., Thimiri Govinda Raj, D.B., Berger, I. (2021). SynBac: Enhanced Baculovirus Genomes by Iterative Recombineering. In: Owens, R.J. (eds) Structural Proteomics. Methods in Molecular Biology, vol 2305. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1406-8_7
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DOI: https://doi.org/10.1007/978-1-0716-1406-8_7
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