A Strategy for the Synthesis of N-Linked Glycoproteins

蛋白质的糖基化在从蛋白质折叠到蛋白质降解的许多过程中起着重要作用。 对癌症等疾病的认识。尽管它的重要性，但人们对它的影响知之甚少 蛋白质糖基化的分子水平。这是由于与隔离相关的困难 同源糖蛋白以下建议将描述一种新的方法， 同源N-连接糖蛋白。这种方法的第一部分涉及进化一种突变的tRNA 合成酶以使突变体tRNA装载氨基酸N-乙酰葡糖胺-天冬酰胺（GlcNAc-Asn） 以便将其整合到体内的蛋白质中。然后，该提案将描述一个 具有增强的转糖基化性质的氨基葡糖苷酶，用于转移复合物 在一些实施方案中，将碳水化合物转化为含有GlcNAc-Asn部分的蛋白质。最后介绍了自动化一锅法的应用 寡糖合成到构建复合碳水化合物底物的突变体 将描述内切氨基葡糖苷酶。如果成功，上述方法的组合将 为高效构建同源N-连接糖蛋白文库提供了新的策略。

Glycosylation of proteins plays a major role in number of processes ranging from protein folding and recognition to diseases such as cancer. Despite its importance there is very little known about the effects of protein glycosylation at the molecular level. This is due to the difficulties associated with isolating homogenous glycoproteins. The following proposal will describe a new approach to the synthesis of homogenous N-linked glycoproteins. The first part of this approach involves evolving a mutant tRNA synthetase to charge a mutant tRNA with the amino acid N-acetyiglucosamine-asparagine (GlcNAc-Asn) in order to incorporate it into proteins in vivo. The proposal will then describe the evolution of a glucosaminidase with enhanced transglycosylation properties, to be used to transfer complex carbohydrates to proteins containing the GlcNAc-Asn moiety. Finally, application of the automated one-pot oligosaccharide synthesis to the construction of a complex carbohydrate substrate for the mutant endoglucosaminidase will be described. If successful the combination of the methods outlined above will provide a new strategy for the efficient construction of libraries of homogenous N-linked glycoproteins.