N-glycosylation mechanism in insect cells

昆虫细胞中的N-糖基化机制

基本信息

批准号：
7850002
负责人：
Donald L. Jarvis
金额：
$ 27.31万
依托单位：
UNIVERSITY OF WYOMING
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-16 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7850002
关键词：
Acylneuraminate Cytidylyltransferase Address Applied Research Baculovirus Expression System Baculoviruses Basic Science Beta-N-Acetylglucosaminidase Bioinformatics Biotechnology Cell Line Cells Chemicals Clinical Confusion Cytidine Monophosphate N-Acetylneuraminic Acid Diagnostic Reagent Engineering Enzymes Eukaryota Genes Genome Glycoproteins Goals Health Human Insect Proteins Insect Vectors Insecta Literature Mediating Metabolic Metabolism Modification Molecular Molecular Cloning Nature Oligosaccharides Pathway interactions Polysaccharides Process Production Protein Engineering Protein Glycosylation Proteins Recombinant Proteins Recombinants Reporting Research Research Personnel Role Scientist Sialic Acids Side Structure System Transgenic Organisms Work cell transformation disease transmission expression vector gene cloning glycosylation human disease improved innovation novel sialic acid permease sialylation therapeutic vaccine

项目摘要

DESCRIPTION (provided by applicant): The baculovirus expression vector system (BEVS) is well known for its ability to produce large amounts of recombinant proteins with eukaryotic modifications. Scientists all over the world have used this system to produce thousands of different recombinant proteins, including many glycoproteins, facilitating research on their structure, function, and roles in human disease. Many biotechnology companies use the BEVS for basic research and/or to produce recombinant proteins for clinical use as vaccines, therapeutics, or diagnostic reagents, and BEVS-manufactured clinical products are clearly on the horizon. However, our relatively poor understanding of insect cell protein processing pathways and their apparent inability to routinely produce recombinant glycoproteins with authentic oligosaccharide side chains are major problems with this system. For the past 19 years, we have studied the molecular mechanisms of protein processing in insect cells. For the past decade, most of our work has focused on protein glycosylation. Through a combination of basic and applied research, we have contributed a better understanding of insect protein /V-glycosylation pathways and produced new insect cell-baculovirus expression systems with improved glycoprotein processing capabilities. In this competing renewal application, we propose to build upon these accomplishments. One general goal will be to extend our previous efforts to metabolically engineer the protein /V-glycosylation pathway of lepidopteran insect cells by transformation with mammalian genes. This is important because additional engineering is needed to produce transgenic insect cell lines with more extensively humanized A/-glycosylation pathways. These cell lines will be broadly useful to biomedical researchers as improved hosts for baculovirus-mediated recombinant /V-glycoprotein production. The other general goal will be to extend our previous efforts to understand the basic nature of the endogenous protein A/-glycosylation pathway in lepidopteran insect cells. We will use metabolic engineering as a powerful and innovative approach to obtain new information on this pathway. We also will continue to clone the genes encoding endogenous protein /V-glycosylation functions and characterize their products. The results will provide a more detailed understanding of insect protein /V-glycosylation mechanisms at the molecular level. This is important because protein /V-glycosylation is a major biosynthetic pathway in all eukaryotes, but it is relatively poorly understood in insect systems. In addition to providing fundamental support for the BEVS, a better understanding of the similarities and differences between insect and mammalian protein /V-glycosylation pathways could reveal ways to interrupt disease transmission by insect vectors or to control agriculturally important insect pests, which would have a major impact on human health.

描述（由申请人提供）：杆状病毒表达矢量系统（BEV）以其能够生产大量具有真核修饰的重组蛋白的能力而闻名。全世界的科学家都使用该系统来生产数千种不同的重组蛋白，包括许多糖蛋白，促进其在人类疾病中的结构，功能和作用的研究。许多生物技术公司使用BEV进行基础研究和/或生产重组蛋白，以临床用作疫苗，疗法或诊断试剂，而BEVS制造的临床产品显然即将上映。但是，我们对昆虫细胞蛋白加工途径的相对较差的理解及其明显无法与正宗的寡糖侧链常规产生重组糖蛋白是该系统的主要问题。在过去的19年中，我们研究了昆虫细胞中蛋白质加工的分子机制。在过去的十年中，我们的大多数工作都集中在蛋白质糖基化上。通过基础研究和应用研究的结合，我们对昆虫蛋白 /V-糖基化途径有了更好的了解，并通过改善了糖蛋白加工能力，产生了新的昆虫细胞 - 巴克洛维病毒表达系统。在此竞争性更新应用中，我们建议以这些成就为基础。一个总体目标是扩展我们以前的努力，以代谢通过用哺乳动物基因转化，通过转化鳞翅目昆虫细胞的蛋白质 /V-糖基化途径。这很重要，因为需要额外的工程来生产具有更广泛人性化的A/ - 糖基化途径的转基因昆虫细胞系。这些细胞系将对生物医学研究人员广泛有用，因为改善了杆状病毒介导的重组 /V-糖蛋白的宿主。另一个一般目标是扩展我们以前的努力，以了解鳞翅目昆虫细胞中内源性蛋白A/ - 糖基化途径的基本性质。我们将使用代谢工程作为一种强大而创新的方法来获取有关此途径的新信息。我们还将继续克隆编码内源性蛋白 /V-糖基化功能并表征其产物的基因。结果将为分子水平的昆虫蛋白 /V-糖基化机制提供更详细的了解。这很重要，因为蛋白质 /V-糖基化是所有真核生物中的主要生物合成途径，但在昆虫系统中相对较少了解。除了为BEV提供基本支持外，对昆虫和哺乳动物蛋白 /V-糖基化途径之间的相似性和差异有了更好的了解，还可以揭示出昆虫向量中断疾病传播或控制农业重要的昆虫的疾病的方法，这可能对人类健康产生重大影响。