Novel Method for in Vitro O-Glycosylation of Proteins: Application for Bioconjugation

Here, we describe a new method for the bioconjugation of a nonglycoprotein with biomolecules. Using polypeptide-α-N-acetylgalactosaminyltransferase II (ppGalNAc-T2), we transfer a C2-modified galactose that has a chemical handle, such as ketone or azide, from its respective UDP-sugars to the Ser/Thr residue(s) of an acceptor polypeptide fused to the nonglycoprotein. The protein with the modified galactose is then coupled to a biomolecule that carries an orthogonal reactive group. As a model system for the nonglycoprotein, we engineered glutathione-S-transferase (GST) protein with a 17-amino-acid-long fusion peptide at the C-terminal end that was expressed as a soluble protein in E. coli. The ppGalNAc-T2 protein, the catalytic domain with the C-terminal lectin domain, was expressed as inclusion bodies in E. coli, and an in vitro folding method was developed to produce milligram quantities of the active enzyme from a liter of bacterial culture. This ppGalNAc-T2 enzyme transfers from the UDP-sugars not only GalNAc but also C2-modified galactose with a chemical handle to the Ser/Thr residue(s) in the fusion peptide. The chemical handle at the C2 of galactose is used for conjugation and assembly of bionanoparticles and preparation of immuno-liposomes for a targeted drug delivery system. This novel method enables one to glycosylate, using ppGalNAc-T2, the important biological nonglycoproteins, such as single-chain antibodies, growth factors, or bacterial toxins, with an engineered 17-residue peptide sequence at the C-terminus of the molecule, for conjugation and coupling.