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

N-连接的糖基化是在真核生物和原核生物中发现的蛋白质修饰。具体地，寡糖从脂质载体转移到特定受体位点基序内的选定天冬酰胺残基。糖组分对于糖蛋白的生物学和物理化学性质至关重要，越来越多的糖蛋白用作重要的治疗剂，如疫苗和抗体。最近在细菌空肠弯曲杆菌中鉴定出一种通用蛋白质N-糖基化系统，并将其功能转移到大肠杆菌中，为体内产生糖蛋白的新方法开辟了道路。已经证明，中心酶，寡糖基转移酶PglB，可以用于将多种结构不同的糖底物从脂质前体转移到蛋白质。由于这种独特的底物灵活性，PglB被认为是用于产生定制糖蛋白的新颖且通用的工具。然而，目前，PglB的应用范围受到限制，因为该酶优选糖基化原核蛋白受体基序D/E-X-N-X-S/T，并且仅在较小程度上糖基化较短的真核形式N-X-S/T。因此，所提出的研究的目标是进化具有宽松的受体基序特异性的PglB变体，其可以用于真核蛋白的位点特异性糖基化。为了允许从大文库中鉴定这种优化的PglB变体，将实施基于噬菌体展示技术的新型筛选方法。

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.