Engineering optimized N-glycosylation in the silkworm silkgland protein expression system

家蚕丝腺蛋白表达系统中的工程优化 N-糖基化

• 批准号: 10380639
• 负责人: Malcolm J. FRASER
• 金额: $ 49.8万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10380639
• 关键词: Address; Affinity Chromatography; Animals; Antibodies; Anticoagulants; Baculoviruses; Biochemistry; Biological Assay; Biomanufacturing; Biomedical Research; Bioreactors; Blood Coagulation Factor; Blood coagulation; Bombyx; Bombyx mori; Carbohydrates; Cell Line; Cells; Cetuximab; Collection; Country; Custom; DNA; Data; Engineering; Enzymes; Erythropoietin; Fiber; Genes; Genetic Engineering; Gland; Glycoengineering; Glycoproteins; Health; Human; Industry; Insecta; Laboratories; Light; Link; Mass Spectrum Analysis; Modeling; Modification; Nickel; Organ; Organism; Pathway interactions; Pattern; Peptides; Polysaccharides; Process; Production; Protein Glycosylation; Proteins; Publications; Recombinant Proteins; Recombinants; Records; Reporting; Research; Research Personnel; Side; Silk; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staphylococcal protein A-sepharose; System; Testing; Therapeutic; Tissue Extracts; Tissues; Transcriptional Regulation; Transgenes; Transgenic Organisms; Trastuzumab; base; clinical application; cost; cost effective; cytokine; design; enzyme activity; expression vector; gene transfer vector; glycosylation; humanized antibody; humanized monoclonal antibodies; interest; novel; promoter; protein expression; skills; success; vector

PROJECT SUMMARY Many biomedically significant proteins, including antibodies, cytokines, anticoagulants, blood clotting factors, and others are glycoproteins. Thus, there is a high demand for systems that can be used to produce recombinant glycoproteins for basic biomedical research and direct clinical applications. Unfortunately, few currently available recombinant protein production systems can produce higher eukaryotic glycoproteins with authentic, relatively homogeneous carbohydrate side chains at relatively low cost. The long-term objective of this proposal is to genetically engineer the silkworm, Bombyx mori, as a system that can fulfill these requirements for recombinant glycoprotein production. Numerous studies have shown that the silkworm silk gland, which has evolved for millions of years as a highly efficient silk protein production and secretion organ, can be engineered to efficiently produce and secrete recombinant proteins. However, transgenic silkworms have not yet been effectively used for recombinant glycoprotein production because the endogenous protein glycosylation pathways of the silk gland cannot properly glycosylate foreign, higher eukaryotic glycoproteins. The proposed research seeks to develop the silkworm as a novel system for recombinant human glycoprotein production by creating transgenic silkworms encoding a set of higher eukaryotic enzymes needed to “humanize” the native silk gland protein N-glycosylation pathway and recombinant human N-glycoproteins of interest. Each transgene will be placed under the control of the tissue-specific Ser1 promoter to target its expression to the middle silk gland. To our knowledge, there is only one prior report of the effective genetic engineering of a protein glycosylation pathway in any multicellular animal, including B. mori. We will build upon our initial success of glycoengineering the protein N-glycosylation pathway of B. mori to significantly advance the use of the silk gland as a bioreactor for recombinant glycoprotein production and secretion. This will have a net positive effect on silk industries in both developed and underdeveloped countries worldwide, allowing value added products important for human health to be produced in an economically feasible manner in addition to the basic silk fiber, thereby significantly increasing the value of this important biomanufacturing platform. The Jarvis and Fraser laboratories have a demonstrated ability to perform this research as shown by their publication records. In addition, they have been productively collaborating on related projects for the past 15 years, generating a significant amount of relevant preliminary data. The complementary skills available in these two labs, their established working relationship, and the preliminary data obtained to date strongly suggest the proposed research can be successfully completed.

项目概要 许多具有生物医学意义的蛋白质，包括抗体、细胞因子、抗凝剂、凝血剂 因子，其他的是糖蛋白。因此，对可用于生产的系统有很高的需求 用于基础生物医学研究和直接临床应用的重组糖蛋白。不幸的是，很少有 目前可用的重组蛋白生产系统可以生产高等真核糖蛋白 真实、相对均质的碳水化合物侧链，成本相对较低。长期目标 该提案是对家蚕（Bombyx mori）进行基因改造，作为一个可以实现这些目标的系统 重组糖蛋白生产的要求。大量研究表明，蚕丝 腺体已经进化了数百万年，是一种高效的丝蛋白生产和分泌器官， 可以被工程化以有效地生产和分泌重组蛋白。然而，转基因蚕 尚未有效地用于重组糖蛋白生产，因为内源蛋白 丝腺的糖基化途径不能正确地糖基化外来的高等真核糖蛋白。 拟议的研究旨在将蚕开发为重组人类的新系统 通过创建编码一组所需的高等真核酶的转基因蚕来生产糖蛋白 将天然丝腺蛋白 N-糖基化途径和重组人 N-糖蛋白“人性化” 兴趣。每个转基因都将置于组织特异性 Ser1 启动子的控制下，以靶向其 表达到中丝腺。 据我们所知，之前只有一篇关于蛋白质的有效基因工程的报道 任何多细胞动物（包括家蚕）的糖基化途径。我们将在初步成功的基础上再接再厉 对家蚕的蛋白质 N-糖基化途径进行糖工程改造，显着促进丝的利用 腺体作为重组糖蛋白生产和分泌的生物反应器。这将产生净积极影响 世界范围内发达国家和不发达国家的丝绸工业，允许增值产品 除了基本的丝绸之外，以经济可行的方式生产对人类健康很重要的产品 纤维，从而显着增加这一重要生物制造平台的价值。 贾维斯和弗雷泽实验室已证明有能力进行这项研究，如他们的研究所示 出版记录。此外，过去 15 年来，他们一直在相关项目上进行富有成效的合作 年，产生了大量相关的初步数据。这些方面可用的补充技能 两个实验室、他们建立的工作关系以及迄今为止获得的初步数据强烈表明 拟议的研究可以顺利完成。