Relocation of the attTn7 Transgene Insertion Site in Bacmid DNA Enhances Baculovirus Genome Stability and Recombinant Protein Expression in Insect Cells
| Autor: | Carissa Grose, Herman E Breukink, Corinne Geertsema, Monique M. van Oers, Tessy A H Hick, Sandra R Abbo, Gorben P. Pijlman, Dominic Esposito, Robin van den Braak, Dirk E. Martens |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Bio Process Engineering Insecta viruses lcsh:QR1-502 Laboratory of Virology Sf9 lcsh:Microbiology chemistry.chemical_compound bioreactor 0302 clinical medicine Bioreactors baculovirus Protein biosynthesis Sf9 Cells 030212 general & internal medicine Replicon Transgenes Homologous Recombination Expression vector PE&RC Recombinant Proteins Cell biology Infectious Diseases insect cells Genetic Engineering Baculoviridae Transgene Genetic Vectors Genome Viral Biology Article Genomic Instability Genome engineering Cell Line Laboratorium voor Virologie Tn7 03 medical and health sciences Virology Animals VLPs Vaccines Virus-Like Particle bacmid Gene protein expression VLAG chikungunya virus Mutagenesis Insertional 030104 developmental biology chemistry EPS DNA genome stability |
| Zdroj: | Viruses 12 (2020) 12 Viruses Volume 12 Issue 12 Viruses, 12(12) Viruses, Vol 12, Iss 1448, p 1448 (2020) |
| ISSN: | 1999-4915 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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