Show simple item record

dc.contributor.authorWilke, Sonja
dc.contributor.authorGroebe, Lothar
dc.contributor.authorMaffenbeier, Vitali
dc.contributor.authorJäger, Volker
dc.contributor.authorGossen, Manfred
dc.contributor.authorJosewski, Jörn
dc.contributor.authorDuda, Agathe
dc.contributor.authorPolle, Lilia
dc.contributor.authorOwens, Raymond J
dc.contributor.authorWirth, Dagmar
dc.contributor.authorHeinz, Dirk W
dc.contributor.authorvan den Heuvel, Joop
dc.contributor.authorBüssow, Konrad
dc.date.accessioned2012-05-30T08:49:37Zen
dc.date.available2012-05-30T08:49:37Zen
dc.date.issued2011en
dc.identifier.citationStreamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development. 2011, 6 (12):e27829 PLoS ONEen_GB
dc.identifier.issn1932-6203en
dc.identifier.pmid22174749en
dc.identifier.doi10.1371/journal.pone.0027829en
dc.identifier.urihttp://hdl.handle.net/10033/226671en
dc.description.abstractStudying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec3.2.8.1 cells. Using preparative cell sorting, stable, high-expressing GFP 'master' cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP).
dc.language.isoenen
dc.rightsArchived with thanks to PloS oneen_GB
dc.subject.meshAnimalsen_GB
dc.subject.meshBiophysical Phenomenaen_GB
dc.subject.meshCHO Cellsen_GB
dc.subject.meshCell Culture Techniquesen_GB
dc.subject.meshCell Lineen_GB
dc.subject.meshCricetinaeen_GB
dc.subject.meshCricetulusen_GB
dc.subject.meshCrystallizationen_GB
dc.subject.meshDNA Nucleotidyltransferasesen_GB
dc.subject.meshGenetic Vectorsen_GB
dc.subject.meshGlycoproteinsen_GB
dc.subject.meshGlycosylationen_GB
dc.subject.meshGreen Fluorescent Proteinsen_GB
dc.subject.meshHumansen_GB
dc.subject.meshLuminescent Proteinsen_GB
dc.subject.meshProtein Structure, Tertiaryen_GB
dc.subject.meshRecombinant Proteinsen_GB
dc.subject.meshRecombination, Geneticen_GB
dc.titleStreamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development.en
dc.typeArticleen
dc.contributor.departmentDepartment of Molecular Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.en_GB
dc.identifier.journalPloS oneen_GB
refterms.dateFOA2018-06-13T19:45:48Z
html.description.abstractStudying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec3.2.8.1 cells. Using preparative cell sorting, stable, high-expressing GFP 'master' cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP).


Files in this item

Thumbnail
Name:
Wilke et al_final.pdf
Size:
1.875Mb
Format:
PDF
Description:
Open Access publication

This item appears in the following Collection(s)

Show simple item record