Site-Specific Glyco-Tagging of Native Proteins for the Development of Biologicals

Glycosylation is an attractive approach to enhance biological properties of pharmaceutical proteins; however, the precise installation of glycans for structure-function studies remains challenging. Here, we describe a chemoenzymatic methodology for glyco-tagging of proteins by peptidoligase catalyzed modification of the N-terminus of a protein with a synthetic glycopeptide ester having an N-acetyl-glucosamine (GlcNAc) moiety to generate an N-GlcNAc modified protein. The GlcNAc moiety can be elaborated into complex glycans by trans-glycosylation using well-defined sugar oxazolines and mutant forms of endo β-N-acetylglucosaminidases (ENGases). The glyco-tagging methodology makes it possible to modify on-demand therapeutic proteins, including heterologous proteins expressed in E. coli, with diverse glycan structures. As a proof of principle, the N-terminus of interleukin (IL)-18 and interferon (IFN)α-2a was modified by a glycopeptide harboring a complex N-glycan without compromising biological potencies. The glyco-tagging methodology was also used to prepare several glycosylated insulin variants that exhibit reduced oligomerization, aggregation, and fibrillization yet maintained cell signaling properties, which are attractive for the development of insulins with improved shelf-lives. It was found that by employing different peptidoligases, it is possible to modify either the A or both chains of human insulin.