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PLoS One. 2011;6(12):e27829. doi: 10.1371/journal.pone.0027829. Epub 2011 Dec 9.

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

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  • 1Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.

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).

PMID:
22174749
[PubMed - indexed for MEDLINE]
PMCID:
PMC3235087
Free PMC Article
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