2.50
Hdl Handle:
http://hdl.handle.net/10033/603996
Title:
Fast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen.
Authors:
Bleckmann, Maren; Schmelz, Stefan; Schinkowski, Christian; Scrima, Andrea; van den Heuvel, Joop ( 0000-0001-5085-4010 )
Abstract:
Recombinant protein expression often presents a bottleneck for the production of proteins for use in many areas of animal-cell biotechnology. Difficult-to-express proteins require the generation of numerous expression constructs, where popular prokaryotic screening systems often fail to identify expression of multi domain or full-length protein constructs. Post-translational modified mammalian proteins require an alternative host system such as insect cells using the Baculovirus Expression Vector System (BEVS). Unfortunately this is time-, labor- and cost-intensive. It is clearly desirable to find an automated and miniaturized fast multi-sample screening method for protein expression in such systems. With this in mind, in this paper a high-throughput initial expression screening method is described using an automated Microcultivation system in conjunction with fast plasmid based transient transfection in insect cells for the efficient generation of protein constructs. The applicability of the system is demonstrated for the difficult to express Nucleotide-binding Oligomerization Domain-containing protein 2 (NOD2). To enable detection of proper protein expression the rather weak plasmid based expression has been improved by a sensitive inline detection system. Here we present the functionality and application of the sensitive SplitGFP (split green fluorescent protein) detection system in insect cells. The successful expression of constructs is monitored by direct measurement of the fluorescence in the BioLector Microcultivation system. Additionally, we show that the results obtained with our plasmid based SplitGFP protein expression screen correlate directly to the level of soluble protein produced in BEVS. In conclusion our automated SplitGFP screen outlines a sensitive, fast and reliable method reducing the time and costs required for identifying the optimal expression construct prior to large scale protein production in baculovirus infected insect cells. This article is protected by copyright. All rights reserved.
Affiliation:
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Citation:
Fast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen. 2016: Biotechnol. Bioeng.
Journal:
Biotechnology and bioengineering
Issue Date:
23-Feb-2016
URI:
http://hdl.handle.net/10033/603996
DOI:
10.1002/bit.25956
PubMed ID:
26913471
Type:
Article
ISSN:
1097-0290
Appears in Collections:
publications of the research group recombinant protein expression (RPEX)

Full metadata record

DC FieldValue Language
dc.contributor.authorBleckmann, Marenen
dc.contributor.authorSchmelz, Stefanen
dc.contributor.authorSchinkowski, Christianen
dc.contributor.authorScrima, Andreaen
dc.contributor.authorvan den Heuvel, Joopen
dc.date.accessioned2016-03-30T09:42:36Zen
dc.date.available2016-03-30T09:42:36Zen
dc.date.issued2016-02-23en
dc.identifier.citationFast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen. 2016: Biotechnol. Bioeng.en
dc.identifier.issn1097-0290en
dc.identifier.pmid26913471en
dc.identifier.doi10.1002/bit.25956en
dc.identifier.urihttp://hdl.handle.net/10033/603996en
dc.description.abstractRecombinant protein expression often presents a bottleneck for the production of proteins for use in many areas of animal-cell biotechnology. Difficult-to-express proteins require the generation of numerous expression constructs, where popular prokaryotic screening systems often fail to identify expression of multi domain or full-length protein constructs. Post-translational modified mammalian proteins require an alternative host system such as insect cells using the Baculovirus Expression Vector System (BEVS). Unfortunately this is time-, labor- and cost-intensive. It is clearly desirable to find an automated and miniaturized fast multi-sample screening method for protein expression in such systems. With this in mind, in this paper a high-throughput initial expression screening method is described using an automated Microcultivation system in conjunction with fast plasmid based transient transfection in insect cells for the efficient generation of protein constructs. The applicability of the system is demonstrated for the difficult to express Nucleotide-binding Oligomerization Domain-containing protein 2 (NOD2). To enable detection of proper protein expression the rather weak plasmid based expression has been improved by a sensitive inline detection system. Here we present the functionality and application of the sensitive SplitGFP (split green fluorescent protein) detection system in insect cells. The successful expression of constructs is monitored by direct measurement of the fluorescence in the BioLector Microcultivation system. Additionally, we show that the results obtained with our plasmid based SplitGFP protein expression screen correlate directly to the level of soluble protein produced in BEVS. In conclusion our automated SplitGFP screen outlines a sensitive, fast and reliable method reducing the time and costs required for identifying the optimal expression construct prior to large scale protein production in baculovirus infected insect cells. This article is protected by copyright. All rights reserved.en
dc.languageENGen
dc.titleFast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen.en
dc.typeArticleen
dc.contributor.departmentHelmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.en
dc.identifier.journalBiotechnology and bioengineeringen

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