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

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1448
Author(s):  
Gorben P. Pijlman ◽  
Carissa Grose ◽  
Tessy A. H. Hick ◽  
Herman E. Breukink ◽  
Robin van den Braak ◽  
...  

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.

Author(s):  
N. Hemamalini ◽  
S. Ezhilmathi ◽  
A. Angela Mercy

Escherichia coli is the most extensively used organism in recombinant protein production. It has several advantages including a very short life cycle, ease of genetic manipulation and the well-known cell biology etc. which makes E. coli as the perfect host for recombinant protein expression. Despite many advantages, E. coli also have few disadvantages such as coupled transcription and translation and lack of eukaryotic post-translational modifications. These challenges can be overcome by adopting several strategies such as, using different E. coli expression vectors, changing the gene sequence without altering the functional domain, modified E. coli strain usage, changing the culture parameters and co-expression with a molecular chaperone. In this review, we present the level of strategies used to enhance the recombinant protein expression and its stability in E. coli.


2010 ◽  
Vol 149 (2) ◽  
pp. 219-227 ◽  
Author(s):  
A. Usami ◽  
S. Ishiyama ◽  
C. Enomoto ◽  
H. Okazaki ◽  
K. Higuchi ◽  
...  

PLoS ONE ◽  
2016 ◽  
Vol 11 (3) ◽  
pp. e0149424 ◽  
Author(s):  
Maren Bleckmann ◽  
Margitta Schürig ◽  
Fang-Fang Chen ◽  
Zen-Zen Yen ◽  
Nils Lindemann ◽  
...  

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