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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).

Original publication

DOI

10.1371/journal.pone.0027829

Type

Journal article

Journal

PLoS One

Publication Date

2011

Volume

6

Keywords

Animals, Biophysical Phenomena, CHO Cells, Cell Culture Techniques, Cell Line, Cricetinae, Cricetulus, Crystallization, DNA Nucleotidyltransferases, Genetic Vectors, Glycoproteins, Glycosylation, Green Fluorescent Proteins, Humans, Luminescent Proteins, Protein Structure, Tertiary, Recombinant Proteins, Recombination, Genetic