Optimized deglycosylation of glycoproteins by peptide-N4-(N-acetyl-β-glucosaminyl)-asparagine amidase fromFlavobacterium meningosepticum
- 1 July 1990
- journal article
- Published by Springer Nature in Glycoconjugate Journal
- Vol. 7 (4), 279-286
- https://doi.org/10.1007/bf01073372
Abstract
Peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F (PNGase F) fromFlavobacterium meningosepticum is a highly useful enzyme for the structural analysis of N(asparagine)-linked carbohydrate chains derived from glycoproteins. The enzyme was enriched using a published procedure [Tarentino AL, Gomez CM, Plummer TH, Jr (1984) Biochemistry 1985:4665–71; Tarentino AL, Plummer TH, Jr (1987) Methods Enzymol 138:770–78] and further purified by hydrophobic interaction HPLC on a weak hydrophobic TSK-Ether column from which it was eluted by a decreasing gradient of 1.7 M ammonium sulphate in 100 mM sodium phosphate, pH 7.0, containing 5 mM EDTA. To determine the optimal conditions for a complete deglycosylation of glycoproteins by PNGase F, experiments were performed with humanα 1-acid glycoprotein, because the five complex type carbohydrate chains are quite resistant to enzymic hydrolysis. The influence of different detergents on the enzyme reaction was studied. Complete deglycosylation of humanα 1-acid glycoprotein was achieved by the use of 60 mU/ml PNGase F in 0.25 M sodium phosphate buffer, pH 8.6, containing 0.2% (w/v) SDS, 20 mM mercaptoethanol and 0.5% Mega-10.Keywords
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