| Autor: |
Sonja Wilke, Lothar Groebe, Vitali Maffenbeier, Volker Jäger, Manfred Gossen, Jörn Josewski, Agathe Duda, Lilia Polle, Raymond J Owens, Dagmar Wirth, Dirk W Heinz, Joop van den Heuvel, Konrad Büssow |
| Jazyk: |
angličtina |
| Rok vydání: |
2011 |
| Předmět: |
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| Zdroj: |
PLoS ONE, Vol 6, Iss 12, p e27829 (2011) |
| Druh dokumentu: |
article |
| ISSN: |
1932-6203 |
| DOI: |
10.1371/journal.pone.0027829 |
| Popis: |
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). |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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