title: An enzyme-based screening system for the rapid assessment of protein N-glycosylation efficiency in yeast.
abstract: N-Glycosylation efficiency is a key parameter when studying components of the protein N-glycosylation pathway, but was recently also recognized as an important factor in the production of glycosylated proteins. We have developed a novel assay to quantify N-glycosylation efficiency of proteins. This assay is based on the secreted activity of yeast acid phosphatase, the proper folding and hence secretion of which is strongly dependent on its N-glycosylation status. The results show that the reporter yields a quantitative measure for protein N-glycosylation in yeast, which is in good agreement with classically used assay based on protein migration patterns on SDS-PAGE. However, the assay is less laborious and is adaptable to high-throughput screening approaches as exemplified.
authors.affiliation: Department of Biotechnology and Chemical Technology, Aalto University, Aalto FI-00076, Finland alexander.frey@aalto.fi.
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Frey
authors.affiliation: Institute of Microbiology, ETH Zürich, Zürich CH-8093, Switzerland.
authors.forename: Markus
authors.initials: M
authors.lastname: Aebi
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism*; D006031:Glycosylation; D057166:High-Throughput Screening Assays / Q000379:methods*; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D000135:Acid Phosphatase; C488916:PHO5 protein, S cerevisiae
keywords: lipid-linked oligosaccharide; oligosaccharyltransferase; protein N-glycosylation; reporter assay; yeast acid phosphatase