Construction and analysis of a novel peptide tag containing an unnatural N-glycosylation site.

The addition of N-glycans to clinically used proteins enhances their therapeutic features. Here we report the design of a novel peptide tag with an unnatural N-glycosylation site, which may increase the N-glycan content of generally any protein. The designed GlycoTags were attached to A1AT, EPO and AGP and constructs were expressed in HEK293 or CHO cells. Hereby we could prove that the attached unnatural N-glycosylation site is decorated with complex-type N-glycans and that the spacer as well as the C-terminal "tail" sequence are critical for the usage of the novel N-glycosylation site. This demonstrates that the novel GlycoTag is a convenient tool to provide proteins with extra N-glycan moieties by simply adding a peptide tag sequence as small as 22 amino acids.