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的许多过程中起着重要作用。尽管它很重要，但在分子水平上对蛋白质糖基化的影响知之甚少。这是由于与分离均质糖蛋白相关的困难。下面的提议将描述一种新的方法来合成均匀的n -连接糖蛋白。该方法的第一部分涉及进化突变tRNA合成酶，使突变tRNA与氨基酸n -乙酰氨基葡萄糖-天冬酰胺（GlcNAc-Asn）结合，以便将其整合到体内蛋白质中。然后，该提案将描述具有增强的转糖基化特性的氨基葡萄糖酶的进化，用于将复杂的碳水化合物转化为含有GlcNAc-Asn片段的蛋白质。最后，将描述自动一锅低聚糖合成在构建突变型内切氨基葡萄糖酶的复杂碳水化合物底物中的应用。如果成功的话，上述方法的结合将为高效构建均质n -连接糖蛋白文库提供一种新的策略。