Engineered yeast with PNGase F on cell surface for releasing of N-glycans from glycoproteins

Peptide-N-(N-acetyl-β-glucosaminyl) asparagine amidase F (PNGase F, EC 3.5.1.52) removes glycan moieties from glycoproteins under relatively mild conditions. This eznyme has been used as a powerful tool in analyzing the structural and biological functions of N-linked glycans of glycoproteins. The PNGase F gene from Flavobacterium meningosepticum was cloned into plasmid pYD1, which enabled regulated expression, secretion and detection. The expression of PNGase F gene at extracellular surface of Saccharomyces cerevisiae was confirmed by immunofluorescence microscopy. Fluorescence activated cell sorter analysis indicated that, after 36 h cultivation, 47.6% of the cell surface was anchored with target proteins. The surface engineered enzyme was confirmed to be active and reached its highest level after induced for 36 h. HPLC analysis showed that the specific activity of the surface-displayed PNGase F was about 12 U/g (cell dry weight).