N-Linked Glycosylation of Antibody Fragments in Escherichia coli

• Published in: Bioconjugate chemistry Vol. 22; no. 3; pp. 488 - 496
• Main Authors: Lizak, Christian, Fan, Yao-Yun, Weber, Thomas Christian, Aebi, Markus
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 16.03.2011
• Subjects: Amino Acid Sequence; Analytical chemistry; Animals; Antigens - immunology; E coli; Escherichia coli - metabolism; Glycoproteins; Glycoproteins - immunology; Glycoproteins - metabolism; Glycosylation; Gram-positive bacteria; Humans; Immunoglobulins; Kinetics; Male; Mice; Models, Molecular; Molecular Sequence Data; Nitrogen - metabolism; Polysaccharides - metabolism; Protein Conformation; Protein Engineering; Single-Chain Antibodies - chemistry; Single-Chain Antibodies - genetics; Single-Chain Antibodies - metabolism; Single-Chain Antibodies - pharmacokinetics; Solubility
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/bc100511k

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.