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.

The Sweet Tooth of Bacteria: Common Themes in Bacterial Glycoconjugates

“…This technology is extremely interesting for industrial applications such as the amelioration of food quality by the engineering of EPS structures or the production of engineered glycoproteins, such as vaccines and human therapeutic agents (e.g., insulin) (131,(559)(560)(561)(562)(563)(564)(565)(566)(567)(568). A better knowledge of the glycosylation mechanisms in beneficial bacteria could cause a revolution in the biosynthesis of therapeutic molecules in prokaryotic vectors.…”

Protein Folding in the Endoplasmic Reticulum

“…The introduction of glycosylation sites into a protein through mutation frequently stabilizes a protein by destabilizing the unfolded state thereby coaxing the protein toward the folded state (Hanson et al 2009). The transferase reaction requires flexibility in the sequon, as the Thr/Ser residue in position 3, is required to loop around to make the Asn nucleophilic for efficient transfer (Helenius and Aebi 2004;Lizak et al 2011). This requirement favors the modification of flexible exposed regions of a protein.…”

Bacterial Protein N-Glycosylation: New Perspectives and Applications

“…Using the bacterial system, Lizak et al (80) demonstrated that introduction of two N-glycan acceptor sites and subsequent N-glycosylation of a singlechain antibody (scFv 3D5) with the C. jejuni N-glycan in E. coli significantly increased the proteolytic stability and solubility without influencing the affinity and serum stability of the single-chain antibody. However, N-glycosylation did not affect the overall clearance rate in the murine model system, which was probably due to the properties of the attached C. jejuni N-glycan (80).…”