O-GlyCORTAR protease, recombinantly expressed from E.coli and tagged with His, is an enzyme that relies on O-glycans to cleave proteins carrying mucin-type O-glycans, including sialylated substrates and glycosylated Serine and Threonine residues at the N-terminus. This protease can process various O-glycan structures, such as sialylated core 1 and core 2 configurations, as well as the Tn antigen. Notably, O-GlyCORTAR protease does not cleave terminally modified serine or threonine residues, nor does it act on N-glycosylation sites of glycoproteins.