Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells

The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar...

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Bibliographic Details
Published in:PloS one 2016-03, Vol.11 (3), p.e0149424-e0149424
Main Authors: Bleckmann, Maren, Schürig, Margitta, Chen, Fang-Fang, Yen, Zen-Zen, Lindemann, Nils, Meyer, Steffen, Spehr, Johannes, van den Heuvel, Joop
Format: Article
Language:English
Subjects:
Analysis
Animals
Baculoviridae - genetics
Baculovirus
Baculoviruses
Biology and Life Sciences
Cell Line
Deoxyribonucleic acid
DNA
Expression vectors
Gene expression
Genes, Reporter - genetics
Genetic aspects
Genetic vectors
Genetic Vectors - genetics
Genomes
Green Fluorescent Proteins - genetics
Homology
IE1 protein
Infections
Insect cells
Insecta - genetics
Insects
New combinations
Physiological aspects
Plasmids - genetics
Production processes
Promoter Regions, Genetic - genetics
Promoters
Protein expression
Proteins
Recombinant proteins
Recombinant Proteins - genetics
Research and Analysis Methods
Sf9 Cells
Spodoptera frugiperda
Transcription termination
Transcription, Genetic - genetics
Transcriptional Activation - genetics
Transfection - methods
Trichoplusia ni
Viral infections
Virology
Viruses
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Summary:The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar expression levels for most proteins so far. The transactivation method is a promising approach for protein expression in Sf21 cells. It combines advantages of BEVS and plasmid-based expression by activating strong virus-dependent promoters on a transfected plasmid by baculoviral coinfection. Here, we identified expression elements required for transactivation. Therefore, we designed several vectors comprising different viral promoters or promoter combinations and tested them for eGFP expression using the automated BioLector microcultivation system. Remarkably, only the combination of the very late promoter p10 together with the homologous region 5 (hr5) could boost expression during transactivation. Other elements, like p10 alone or the late viral promoter polH, did not respond to transactivation. A new combination of hr5 and p10 with the strongest immediate early OpMNPV viral promoter OpIE2 improved the yield of eGFP by ~25% in comparison to the previous applied hr5-IE1-p10 expression cassette. Furthermore, we observed a strong influence of the transcription termination sequence and vector backbone on the level of expression. Finally, the expression levels for transactivation, BEVS and solely plasmid-based expression were compared for the marker protein eGFP, underlining the potential of transactivation for fast recombinant protein expression in Sf21 cells. In conclusion, essential elements for transactivation could be identified. The optimal elements were applied to generate an improved vector applicable in virus-free plasmid-based expression, transactivation and BEVS.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0149424