A Strategy for the Synthesis of N-Linked Glycoproteins

N-连接糖蛋白的合成策略

• 批准号: 6885235
• 负责人: Clay Samuel Bennett
• 金额: $ 4.21万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6885235
• 关键词: Escherichia coli; N acetylglucosamine; N acetylglucosaminidase; aminoacid tRNA ligase; asparagine; combinatorial chemistry; directed evolution; genetic library; genetic screening; glycoproteins; glycosylation; method development; mutant; oligosaccharides; peptide chemical synthesis; peptide library; postdoctoral investigator; site directed mutagenesis

DESCRIPTION (provided by applicant): Glycosylation of proteins plays a major role in a 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-acetyglucosamine-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.

描述（由申请人提供）：蛋白质的糖基化在从蛋白质折叠和识别到癌症等疾病等许多过程中起着重要作用。尽管它很重要，但对分子水平蛋白质糖基化的影响鲜为人知。这是由于与分离同质糖蛋白有关的困难。以下建议将描述一种合成同质N连接糖蛋白的新方法。该方法的第一部分涉及将突变型tRNA合成酶进化为与氨基酸N-环葡萄糖胺 - 天冬酰胺（GlcNAC-ASN）充电突变型TRNA，以便将其掺入体内蛋白质中。然后，该提案将描述具有增强的克糖基化特性的葡糖胺酶的演变，用于将复杂的碳水化合物转移到含有GlcNAC-ASN部分的蛋白质中。最后，将描述将自动的单锅寡糖合成在突变体内糖糖胺酶中的复杂碳水化合物底物构造中的应用。如果成功，上面概述的方法的组合将为有效构建同质N连接糖蛋白的库提供新的策略。