For the hydrolase binding experiments, the homogenous fraction of OMC45 polypeptide was first treated with N-glycanase (to remove all N-linked oligosaccharide moieties) and the resulting OMC45 polypeptide was treated with O-glycanase (an endoenzyme known to cleave O-linked OS chains). Both incubations were done overnight at 37°C according to the New England Biolabs’ instructions. After enzymatic digestion, deglycosylated OMC45 polypeptide and native OMC45 polypeptide were conjugated to AminoLink Plus coupling gel (Pierce Chemical Co.) separately at pH 10.0 according to the manufacturer’s instructions. As a control, same units of N-glycanase and O-glycanase were conjugated to AminoLink Plus coupling gel. A centrifugation assay was performed to determine the binding efficacy of hydrolases to the deglysosylated OMC45 polypeptide following the method of Nagdas et al. [13 (link)].
O glycanase
O-glycanase is an enzyme used to remove O-linked glycans from glycoproteins. It functions by cleaving the glycosidic linkage between the carbohydrate and the serine or threonine residue of the polypeptide backbone.
2 protocols using o glycanase
Deglycosylation and Hydrolase Binding Assay of OMC45
O-Glycosidic Linkage Removal from Proteins
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