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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

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 w...

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Autores principales: Pijlman, Gorben P., Grose, Carissa, Hick, Tessy A. H., Breukink, Herman E., van den Braak, Robin, Abbo, Sandra R., Geertsema, Corinne, van Oers, Monique M., Martens, Dirk E., Esposito, Dominic
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7765880/
https://www.ncbi.nlm.nih.gov/pubmed/33339324
http://dx.doi.org/10.3390/v12121448
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author Pijlman, Gorben P.
Grose, Carissa
Hick, Tessy A. H.
Breukink, Herman E.
van den Braak, Robin
Abbo, Sandra R.
Geertsema, Corinne
van Oers, Monique M.
Martens, Dirk E.
Esposito, Dominic
author_facet Pijlman, Gorben P.
Grose, Carissa
Hick, Tessy A. H.
Breukink, Herman E.
van den Braak, Robin
Abbo, Sandra R.
Geertsema, Corinne
van Oers, Monique M.
Martens, Dirk E.
Esposito, Dominic
author_sort Pijlman, Gorben P.
collection PubMed
description 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.
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spelling pubmed-77658802020-12-28 Relocation of the attTn7 Transgene Insertion Site in Bacmid DNA Enhances Baculovirus Genome Stability and Recombinant Protein Expression in Insect Cells Pijlman, Gorben P. Grose, Carissa Hick, Tessy A. H. Breukink, Herman E. van den Braak, Robin Abbo, Sandra R. Geertsema, Corinne van Oers, Monique M. Martens, Dirk E. Esposito, Dominic Viruses Article 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. MDPI 2020-12-16 /pmc/articles/PMC7765880/ /pubmed/33339324 http://dx.doi.org/10.3390/v12121448 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Pijlman, Gorben P.
Grose, Carissa
Hick, Tessy A. H.
Breukink, Herman E.
van den Braak, Robin
Abbo, Sandra R.
Geertsema, Corinne
van Oers, Monique M.
Martens, Dirk E.
Esposito, Dominic
Relocation of the attTn7 Transgene Insertion Site in Bacmid DNA Enhances Baculovirus Genome Stability and Recombinant Protein Expression in Insect Cells
title Relocation of the attTn7 Transgene Insertion Site in Bacmid DNA Enhances Baculovirus Genome Stability and Recombinant Protein Expression in Insect Cells
title_full Relocation of the attTn7 Transgene Insertion Site in Bacmid DNA Enhances Baculovirus Genome Stability and Recombinant Protein Expression in Insect Cells
title_fullStr Relocation of the attTn7 Transgene Insertion Site in Bacmid DNA Enhances Baculovirus Genome Stability and Recombinant Protein Expression in Insect Cells
title_full_unstemmed Relocation of the attTn7 Transgene Insertion Site in Bacmid DNA Enhances Baculovirus Genome Stability and Recombinant Protein Expression in Insect Cells
title_short Relocation of the attTn7 Transgene Insertion Site in Bacmid DNA Enhances Baculovirus Genome Stability and Recombinant Protein Expression in Insect Cells
title_sort relocation of the atttn7 transgene insertion site in bacmid dna enhances baculovirus genome stability and recombinant protein expression in insect cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7765880/
https://www.ncbi.nlm.nih.gov/pubmed/33339324
http://dx.doi.org/10.3390/v12121448
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