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

2.50
Hdl Handle:
http://hdl.handle.net/10033/600982
Title:
Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells.
Authors:
Bleckmann, Maren; Schürig, Margitta; Chen, Fang-Fang; Yen, Zen-Zen; Lindemann, Nils; Meyer, Steffen; Spehr, Johannes; van den Heuvel, Joop ( 0000-0001-5085-4010 )
Abstract:
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.
Affiliation:
Helmholtz Centre for infection research (HZI), Inhoffenstraße 7, 38124 Braunschweig, Germany.
Citation:
Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells. 2016, 11 (3):e0149424 PLoS ONE
Journal:
PloS one
Issue Date:
2016
URI:
http://hdl.handle.net/10033/600982
DOI:
10.1371/journal.pone.0149424
PubMed ID:
26934632
Type:
Article
Language:
en
ISSN:
1932-6203
Appears in Collections:
publications of the research group recombinant protein expression (RPEX)

Full metadata record

DC FieldValue Language
dc.contributor.authorBleckmann, Marenen
dc.contributor.authorSchürig, Margittaen
dc.contributor.authorChen, Fang-Fangen
dc.contributor.authorYen, Zen-Zenen
dc.contributor.authorLindemann, Nilsen
dc.contributor.authorMeyer, Steffenen
dc.contributor.authorSpehr, Johannesen
dc.contributor.authorvan den Heuvel, Joopen
dc.date.accessioned2016-03-09T08:18:20Zen
dc.date.available2016-03-09T08:18:20Zen
dc.date.issued2016en
dc.identifier.citationIdentification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells. 2016, 11 (3):e0149424 PLoS ONEen
dc.identifier.issn1932-6203en
dc.identifier.pmid26934632en
dc.identifier.doi10.1371/journal.pone.0149424en
dc.identifier.urihttp://hdl.handle.net/10033/600982en
dc.description.abstractThe 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.en
dc.language.isoenen
dc.titleIdentification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells.en
dc.typeArticleen
dc.contributor.departmentHelmholtz Centre for infection research (HZI), Inhoffenstraße 7, 38124 Braunschweig, Germany.en
dc.identifier.journalPloS oneen
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