N-glycosylation mechanism in insect cells

描述（由申请人提供）：众所周知，杆状病毒表达载体系统（BEVS）能够产生大量经真核修饰的重组蛋白。世界各地的科学家已经使用该系统生产了数千种不同的重组蛋白，包括许多糖蛋白，促进了对其结构，功能和在人类疾病中的作用的研究。许多生物技术公司使用BEVS进行基础研究和/或生产临床使用的重组蛋白质作为疫苗，治疗剂或诊断试剂，BEVS制造的临床产品显然即将出现。然而，我们对昆虫细胞蛋白质加工途径的了解相对较少，并且它们明显不能常规地产生具有真实寡糖侧链的重组糖蛋白，这是该系统的主要问题。在过去的19年里，我们研究了昆虫细胞中蛋白质加工的分子机制。在过去的十年里，我们的大部分工作都集中在蛋白质糖基化上。通过基础和应用研究的结合，我们对昆虫蛋白/V-糖基化途径有了更好的理解，并产生了具有改进的糖蛋白加工能力的新昆虫细胞-杆状病毒表达系统。在这个竞争性的更新应用程序中，我们建议在这些成就的基础上再接再厉。一个总体目标将是扩展我们先前的努力，通过用哺乳动物基因转化来代谢工程化鳞翅目昆虫细胞的蛋白质/V-糖基化途径。这是重要的，因为需要额外的工程改造来产生具有更广泛的人源化N-糖基化途径的转基因昆虫细胞系。这些细胞系将广泛用于生物医学研究人员作为杆状病毒介导的重组/V-糖蛋白生产的改进宿主。另一个总体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个强大的和创新的方法，以获得关于这一途径的新信息。我们还将继续克隆编码内源性蛋白质/V-糖基化功能的基因并表征其产物。这些结果将在分子水平上提供对昆虫蛋白/V-糖基化机制的更详细的理解。这是重要的，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，但在昆虫系统中了解相对较少。除了为BEVS提供基本支持外，更好地了解昆虫和哺乳动物蛋白质/V-糖基化途径之间的相似性和差异，可以揭示阻断昆虫媒介传播疾病或控制农业重要害虫的方法，这将对人类健康产生重大影响。

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.