Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development.

2.50
Hdl Handle:
http://hdl.handle.net/10033/226671
Title:
Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development.
Authors:
Wilke, Sonja; Groebe, Lothar; Maffenbeier, Vitali; Jäger, Volker; Gossen, Manfred; Josewski, Jörn; Duda, Agathe; Polle, Lilia; Owens, Raymond J; Wirth, Dagmar; Heinz, Dirk W; van den Heuvel, Joop; Büssow, Konrad
Abstract:
Studying 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).
Affiliation:
Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Citation:
Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development. 2011, 6 (12):e27829 PLoS ONE
Journal:
PloS one
Issue Date:
2011
URI:
http://hdl.handle.net/10033/226671
DOI:
10.1371/journal.pone.0027829
PubMed ID:
22174749
Type:
Article
Language:
en
ISSN:
1932-6203
Appears in Collections:
Publications from Division of Molekulare Struktur Biologie (MOSB)

Full metadata record

DC FieldValue Language
dc.contributor.authorWilke, Sonjaen_GB
dc.contributor.authorGroebe, Lotharen_GB
dc.contributor.authorMaffenbeier, Vitalien_GB
dc.contributor.authorJäger, Volkeren_GB
dc.contributor.authorGossen, Manfreden_GB
dc.contributor.authorJosewski, Jörnen_GB
dc.contributor.authorDuda, Agatheen_GB
dc.contributor.authorPolle, Liliaen_GB
dc.contributor.authorOwens, Raymond Jen_GB
dc.contributor.authorWirth, Dagmaren_GB
dc.contributor.authorHeinz, Dirk Wen_GB
dc.contributor.authorvan den Heuvel, Joopen_GB
dc.contributor.authorBüssow, Konraden_GB
dc.date.accessioned2012-05-30T08:49:37Z-
dc.date.available2012-05-30T08:49:37Z-
dc.date.issued2011-
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.issn1932-6203-
dc.identifier.pmid22174749-
dc.identifier.doi10.1371/journal.pone.0027829-
dc.identifier.urihttp://hdl.handle.net/10033/226671-
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).en_GB
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

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