Evolvierung einer bakteriellen Oligosaccharyltransferase (PglB) für die sequenzspezifische Glykosylierung eukaryotischer Proteine in E. coli

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N-linked glycosylation is a protein modification found in both eukaryotes and prokaryotes. Specifically, an oligosaccharide gets transferred from a lipid carrier to selected asparagine residues within a particular acceptor site motif. The sugar component is of fundamental importance for the biological and physicochemical properties of glycoproteins, a growing number of which serve as important therapeutics such as vaccines and antibodies. The recent identification of a general protein N-glycosylation system in the bacterium Campylobacter jejuni and its functional transfer to Escherichia coli opened the way for novel approaches to produce glycoproteins in vivo. It was demonstrated that the central enzyme, the oligosaccharyltransferase PglB, can be exploited to transfer a wide array of structurally different sugar substrates from a lipid precursor to proteins. Due to this unique substrate flexibility, PglB was suggested as novel and versatile tool for the generation of custom glycoproteins. At present, however, the scope of application of PglB is limited since the enzyme glycosylates preferably the prokaryotic protein acceptor motif D/E-X-N-X-S/T and only to a minor extent the shorter eukaryotic version N-X-S/T. The goal of the proposed research is therefore to evolve a PglB variant with a relaxed acceptor motif specificity which can be exploited for the site-specific glycosylation of eukaryotic proteins. To allow for the identification of such an optimized PglB variant out of a large library, a novel screening method based on phage display technology will be implemented.