2.50
Hdl Handle:
http://hdl.handle.net/10033/603923
Title:
Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda.
Authors:
Bleckmann, Maren; Fritz, Markus H-Y; Bhuju, Sabin; Jarek, Michael; Schürig, Margitta; Geffers, Robert ( 0000-0003-4409-016X ) ; Benes, Vladimir; Besir, Hüseyin; van den Heuvel, Joop ( 0000-0001-5085-4010 )
Abstract:
The Baculoviral Expression Vector System (BEVS) is the most commonly used method for high expression of recombinant protein in insect cells. Nevertheless, expression of some target proteins--especially those entering the secretory pathway--provides a severe challenge for the baculovirus infected insect cells, due to the reorganisation of intracellular compounds upon viral infection. Therefore, alternative strategies for recombinant protein production in insect cells like transient plasmid-based expression or stable expression cell lines are becoming more popular. However, the major bottleneck of these systems is the lack of strong endogenous polymerase II dependent promoters, as the strong baculoviral p10 and polH promoters used in BEVS are only functional in presence of the viral transcription machinery during the late phase of infection. In this work we present a draft genome and a transcriptome analysis of Sf21 cells for the identification of the first known endogenous Spodoptera frugiperda promoters. Therefore, putative promoter sequences were identified and selected because of high mRNA level or in analogy to other strong promoters in other eukaryotic organism. The chosen endogenous Sf21 promoters were compared to early viral promoters for their efficiency to trigger eGFP expression using transient plasmid based transfection in a BioLector Microfermentation system. Furthermore, promoter activity was not only shown in Sf21 cells but also in Hi5 cells. The novel endogenous Sf21 promoters were ranked according to their activity and expand the small pool of available promoters for stable insect cell line development and transient plasmid expression in insect cells. The best promoter was used to improve plasmid based transient transfection in insect cells substantially.
Affiliation:
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Citation:
Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda. 2015, 10 (8):e0132898 PLoS ONE
Journal:
PloS one
Issue Date:
2015
URI:
http://hdl.handle.net/10033/603923
DOI:
10.1371/journal.pone.0132898
PubMed ID:
26263512
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.authorFritz, Markus H-Yen
dc.contributor.authorBhuju, Sabinen
dc.contributor.authorJarek, Michaelen
dc.contributor.authorSchürig, Margittaen
dc.contributor.authorGeffers, Roberten
dc.contributor.authorBenes, Vladimiren
dc.contributor.authorBesir, Hüseyinen
dc.contributor.authorvan den Heuvel, Joopen
dc.date.accessioned2016-03-29T14:34:28Zen
dc.date.available2016-03-29T14:34:28Zen
dc.date.issued2015en
dc.identifier.citationGenomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda. 2015, 10 (8):e0132898 PLoS ONEen
dc.identifier.issn1932-6203en
dc.identifier.pmid26263512en
dc.identifier.doi10.1371/journal.pone.0132898en
dc.identifier.urihttp://hdl.handle.net/10033/603923en
dc.description.abstractThe Baculoviral Expression Vector System (BEVS) is the most commonly used method for high expression of recombinant protein in insect cells. Nevertheless, expression of some target proteins--especially those entering the secretory pathway--provides a severe challenge for the baculovirus infected insect cells, due to the reorganisation of intracellular compounds upon viral infection. Therefore, alternative strategies for recombinant protein production in insect cells like transient plasmid-based expression or stable expression cell lines are becoming more popular. However, the major bottleneck of these systems is the lack of strong endogenous polymerase II dependent promoters, as the strong baculoviral p10 and polH promoters used in BEVS are only functional in presence of the viral transcription machinery during the late phase of infection. In this work we present a draft genome and a transcriptome analysis of Sf21 cells for the identification of the first known endogenous Spodoptera frugiperda promoters. Therefore, putative promoter sequences were identified and selected because of high mRNA level or in analogy to other strong promoters in other eukaryotic organism. The chosen endogenous Sf21 promoters were compared to early viral promoters for their efficiency to trigger eGFP expression using transient plasmid based transfection in a BioLector Microfermentation system. Furthermore, promoter activity was not only shown in Sf21 cells but also in Hi5 cells. The novel endogenous Sf21 promoters were ranked according to their activity and expand the small pool of available promoters for stable insect cell line development and transient plasmid expression in insect cells. The best promoter was used to improve plasmid based transient transfection in insect cells substantially.en
dc.language.isoenen
dc.titleGenomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda.en
dc.typeArticleen
dc.contributor.departmentHelmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.en
dc.identifier.journalPloS oneen

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