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.

Keywords: lipid-linked oligosaccharide; oligosaccharyltransferase; protein N-glycosylation; reporter assay; yeast acid phosphatase.