title: Novel method for in vitro O-glycosylation of proteins: application for bioconjugation.
abstract: Here, we describe a new method for the bioconjugation of a nonglycoprotein with biomolecules. Using polypeptide-alpha- 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.
authors.affiliation: Structural Glycobiology Section, and Basic Research Program, SAIC-Frederick Inc, Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 21702 , USA.
authors.forename: Boopathy
authors.initials: B
authors.lastname: Ramakrishnan
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Boeggeman
authors.forename: Pradman K
authors.initials: PK
authors.lastname: Qasba
mesh_terms: D002384:Catalysis; D017930:Genes, Reporter / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D002479:Inclusion Bodies; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D011506:Proteins / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D011506:Proteins