Recombinant Glycans on an S-Layer Self-Assembly Protein: A New Dimension for Nanopatterned Biomaterials

Crucial biological phenomena are mediated through carbohydrates that are displayed in a defined manner and interact with molecular scale precision. We lay the groundwork for the integration of recombinant carbohydrates into a “biomolecular construction kit” for the design of new biomaterials, by utilizing the self‐assembly system of the crystalline cell surface (S)‐layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a. SgsE is a naturally O‐glycosylated protein, with intrinsic properties that allow it to function as a nanopatterned matrix for the periodic display of glycans. By using a combined carbohydrate/protein engineering approach, two types of S‐layer neoglycoproteins are produced in Escherichia coli. Based on the identification of a suitable periplasmic targeting system for the SgsE self‐assembly protein as a cellular prerequisite for protein glycosylation, and on engineering of one of the natural protein O‐glycosylation sites into a target for N‐glycosylation, the heptasaccharide from the AcrA protein of Campylobacter jejuni and the O7 polysaccharide of E. coli are co‐ or post‐translationally transferred to the S‐layer protein by the action of the oligosaccharyltransferase PglB. The degree of glycosylation of the S‐layer neoglycoproteins after purification from the periplasmic fraction reaches completeness. Electron microscopy reveals that recombinant glycosylation is fully compatible with the S‐layer protein self‐assembly system. Tailor‐made (“functional”) nanopatterned, self‐assembling neoglycoproteins may open up new strategies for influencing and controlling complex biological systems with potential applications in the areas of biomimetics, drug targeting, vaccine design, or diagnostics. Nanopatterned neoglycoproteins are produced by a combined protein/carbohydrate engineering approach from the self‐assembling cell‐surface layer (S‐layer) protein SgsE of Geobacillus stearothermophilus and a heptasaccharide from Campylobacter jejuni and the Escherichia coli O7 antigen. The S‐layer matrix assures a controllable, periodic and nanometer‐scale display of the glycans as a base for the conceptuation of novel biomaterials, which have incorporated carbohydrate‐mediated functions.