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Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development.
Wilke, Sonja Groebe, Lothar Maffenbeier, Vitali Jäger, Volker Gossen, Manfred Josewski, Jörn Duda, Agathe Polle, Lilia Owens, Raymond J Wirth, Dagmar
Wilke, Sonja
Groebe, Lothar
Maffenbeier, Vitali
Jäger, Volker
Gossen, Manfred
Josewski, Jörn
Duda, Agathe
Polle, Lilia
Owens, Raymond J
Wirth, Dagmar
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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).
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Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development. 2011, 6 (12):e27829 PLoS ONE
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en
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1932-6203
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