Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland.
LimmaTech Biologics AG, Schlieren, Switzerland.
Metab Eng. 2017 Nov;44:293-301. doi: 10.1016/j.ymben.2017.10.012. Epub 2017 Nov 1.
Polysialic acid (polySia) is a posttranslational modification found on only a handful of proteins in the central nervous and immune systems. The addition of polySia to therapeutic proteins improves pharmacokinetics and reduces immunogenicity. To date, polysialylation of therapeutic proteins has only been achieved in vitro by chemical or chemoenzymatic strategies. In this work, we develop a biosynthetic pathway for site-specific polysialylation of recombinant proteins in the cytoplasm of Escherichia coli. The pathway takes advantage of a bacterial cytoplasmic polypeptide-glycosyltransferase to establish a site-specific primer on the target protein. The glucose primer is extended by glycosyltransferases derived from lipooligosaccharide, lipopolysaccharide and capsular polysaccharide biosynthesis from different bacterial species to synthesize long chain polySia. We demonstrate the new biosynthetic route by modifying green fluorescent proteins and a therapeutic DARPin (designed ankyrin repeat protein).
聚唾液酸（polySia）是一种仅存在于中枢神经系统和免疫系统少数几种蛋白质上的翻译后修饰。在治疗性蛋白上添加聚唾液酸可以改善药代动力学并降低免疫原性。迄今为止，治疗性蛋白的聚唾液酸化仅通过化学或化学酶策略在体外实现。在这项工作中，我们开发了一种在大肠杆菌细胞质中对重组蛋白进行定点聚唾液酸化的生物合成途径。该途径利用细菌细胞质多肽糖基转移酶在靶蛋白上建立一个定点引物。葡萄糖引物通过来自不同细菌物种的脂寡糖、脂多糖和荚膜多糖生物合成的糖基转移酶延伸，以合成长链聚唾液酸。我们通过修饰绿色荧光蛋白和一种治疗性 DARPin（设计的锚蛋白重复蛋白）来证明新的生物合成途径。
