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).
Highlights
Structural and biophysical studies of glycosylated proteins require recombinant protein samples of high quality and homogeneity
Glycosylation mutant recombinase-mediated cassette exchange (RMCE) master cell lines Stable RMCE master cell lines were derived from the CHO
When GFP is exchanged by Flpmediated recombination against a cassette including a promoter and start codon, deleted neomycin phosphotransferase (Dneo) becomes complemented and the cell becomes resistant against the antibiotic G418, allowing for selection of recombinant cells
Summary
Structural and biophysical studies of glycosylated proteins require recombinant protein samples of high quality and homogeneity. Production of glycoproteins relies mostly on eukaryotic protein expression systems [1]. Protein-linked glycan chains are essential for protein folding and secretion, but they cause sample heterogeneity, which complicates protein crystallization and biophysical measurements, e.g. determination of molecular mass and oligomerization status [2]. Inhibitors and mutations of N-acetylglucosaminyl-transferase I (GnTI) prevent the processing of N-linked glycans beyond the high-mannose type, leading to smaller and more homogeneous modifications [3,4]. High-mannose type glycans can be truncated efficiently to a single N-acetylglucosamine by endoglycosidase H, which usually does not affect protein stability, but often improves crystal growth [4,8]
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