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Article

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

1
Laboratory of Virology, Wageningen University, Droevendaalsesteeg 1, 6708PB Wageningen, The Netherlands
2
Protein Expression Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702, USA
3
Bioprocess Engineering, Wageningen University, Droevendaalsesteeg 1, 6708PB Wageningen, The Netherlands
*
Author to whom correspondence should be addressed.
Academic Editors: Carla Varanda and Patrick Materatski
Viruses 2020, 12(12), 1448; https://doi.org/10.3390/v12121448
Received: 23 November 2020 / Revised: 9 December 2020 / Accepted: 14 December 2020 / Published: 16 December 2020
(This article belongs to the Special Issue The Application of Viruses to Biotechnology)
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. View Full-Text
Keywords: baculovirus; insect cells; bacmid; Tn7; genome stability; protein expression; chikungunya virus; VLPs; bioreactor baculovirus; insect cells; bacmid; Tn7; genome stability; protein expression; chikungunya virus; VLPs; bioreactor
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MDPI and ACS Style

Pijlman, G.P.; Grose, C.; Hick, T.A.H.; Breukink, H.E.; van den Braak, R.; Abbo, S.R.; Geertsema, C.; van Oers, M.M.; Martens, D.E.; Esposito, D. Relocation of the attTn7 Transgene Insertion Site in Bacmid DNA Enhances Baculovirus Genome Stability and Recombinant Protein Expression in Insect Cells. Viruses 2020, 12, 1448. https://doi.org/10.3390/v12121448

AMA Style

Pijlman GP, Grose C, Hick TAH, Breukink HE, van den Braak R, Abbo SR, Geertsema C, van Oers MM, Martens DE, Esposito D. Relocation of the attTn7 Transgene Insertion Site in Bacmid DNA Enhances Baculovirus Genome Stability and Recombinant Protein Expression in Insect Cells. Viruses. 2020; 12(12):1448. https://doi.org/10.3390/v12121448

Chicago/Turabian Style

Pijlman, Gorben P., Carissa Grose, Tessy A. H. Hick, Herman E. Breukink, Robin van den Braak, Sandra R. Abbo, Corinne Geertsema, Monique M. van Oers, Dirk E. Martens, and Dominic Esposito. 2020. "Relocation of the attTn7 Transgene Insertion Site in Bacmid DNA Enhances Baculovirus Genome Stability and Recombinant Protein Expression in Insect Cells" Viruses 12, no. 12: 1448. https://doi.org/10.3390/v12121448

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