Abstract
N-glycosylation is a common posttranslational modification of proteins in eukaryotic cells. The modification is often analyzed in cells which are able to produce extracellular, glycosylated proteins. Here we report an improved method of the use of genetically modified, secreted alkaline phosphatase (SEAP) as a reporter glycoprotein which may be used for glycoanalysis. Additional N-glycosylation sites introduced by site-directed mutagenesis significantly increased secretion of the protein. An improved purification protocol of recombinant SEAP from serum or serum-free media is also proposed. The method enables fast and efficient separation of reporter glycoprotein from a relatively small amount of medium (0.5-10 ml) with a high recovery level. As a result, purified SEAP was ready for enzymatic de-glycosylation without buffer exchange, sample volume reductions or other procedures, which are usually time-consuming and may cause partial loss of the reporter glycoprotein.
Highlights
In the majority of eukaryotic cells, glycosylation is among the most frequent posttranslational modifications of macromolecules, i.e., glycoproteins, proteoglycans, and lipids
In eukaryotic tissues most N-glycans are converted from initial, high mannose type to mature forms, containing additional N-acetylglucosamine, galactose, fucose and sialic acid residues
The main goal of this project was to design a new version of the glycoprotein which: 1) may be synthesized and secreted to the culture media by transiently transfected mammalian cells of different types at higher rates than previously reported; 2) the secretion level may be monitored in seconds or minutes using simple colorimetric assay; 3) the purification procedure is fast, efficient, and is not dependent on type of serum and culture media; 4) the reporter protein possesses at least three functional glycosylation sites, in contrast to the original secreted alkaline phosphatase (SEAP), which contains only one functional N-glycosylation site [17, 18]
Summary
In the majority of eukaryotic cells, glycosylation is among the most frequent posttranslational modifications of macromolecules, i.e., glycoproteins, proteoglycans, and lipids. Among different types of protein posttranslational modifications, N-glycosylation is one of the most common. This modification is dependent on specific three-amino-acid motifs in glycoproteins (sequons). The sequon for N-glycosylation is either N-X-S or N-X-T, where X is any amino acid except proline. Especially those with high activity of secretory pathways (ER-Golgi processing), most proteins are subjected to N-glycosylation [2]. In eukaryotic tissues most N-glycans are converted from initial, high mannose type to mature forms, containing additional N-acetylglucosamine, galactose, fucose and sialic acid residues. A detailed analysis of N-glycans covalently attached to glycoproteins of mammalian cell
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