title: N-Linked glycosylation of antibody fragments in Escherichia coli.
abstract: Glycosylation is the predominant protein modification to diversify the functionality of proteins. In particular, N-linked protein glycosylation can increase the biophysical and pharmacokinetic properties of therapeutic proteins. However, the major challenges in studying the consequences of protein glycosylation on a molecular level are caused by glycan heterogeneities of currently used eukaryotic expression systems, but the discovery of the N-linked protein glycosylation system in the ε-proteobacterium Campylobacter jejuni and its functional transfer to Escherichia coli opened up the possibility to produce glycoproteins in bacteria. Toward this goal, we elucidated whether antibody fragments, a potential class of therapeutic proteins, are amenable to bacterial N-linked glycosylation, thereby improving their biophysical properties. We describe a new strategy for glycoengineering and production of quantitative amounts of glycosylated scFv 3D5 at high purity. The analysis revealed the presence of a homogeneous N-glycan that significantly increased the stability and the solubility of the 3D5 antibody fragment. The process of bacterial N-linked glycosylation offers the possibility to specifically address and alter the biophysical properties of proteins.
authors.affiliation: Department of Biology, Institute of Microbiology, ETH Zürich, CH-8093 Zürich, Switzerland.
authors.forename: Christian
authors.initials: C
authors.lastname: Lizak
authors.forename: Yao-Yun
authors.initials: YY
authors.lastname: Fan
authors.forename: Thomas Christian
authors.initials: TC
authors.lastname: Weber
authors.forename: Markus
authors.initials: M
authors.lastname: Aebi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000276:immunology; D004926:Escherichia coli / Q000378:metabolism*; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008297:Male; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000493:pharmacokinetics; D012995:Solubility
chemical_list: D000941:Antigens; D006023:Glycoproteins; D011134:Polysaccharides; D057127:Single-Chain Antibodies; D009584:Nitrogen