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

Authors :
Gorben P. Pijlman
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
Dominic Esposito
Source :
Viruses, Vol 12, Iss 12, p 1448 (2020)
Publication Year :
2020
Publisher :
MDPI AG, 2020.

Abstract

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.

Details

Language :
English
ISSN :
19994915
Volume :
12
Issue :
12
Database :
Directory of Open Access Journals
Journal :
Viruses
Publication Type :
Academic Journal
Accession number :
edsdoj.8500244a4580486199fd2a92c3836476
Document Type :
article
Full Text :
https://doi.org/10.3390/v12121448