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: 9982365
• 负责人: Malcolm J. FRASER
• 金额: $ 51.16万
• 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 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9982365
• 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; 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.

项目摘要 许多具有生物医学意义的蛋白质，倾斜抗体，细胞因子，抗凝剂，血液凝结 因素，其他因素是糖蛋白。 不幸的是，重组糖蛋白用于Baskic生物医学研究和直接临床应用 目前可用的重组蛋白蛋白生产系统可以与 真实的，相对均匀的碳水化合物面，相对低成本。 该提议是为了肯定地设计蚕（Bombyx Mori），如可以满足这些的astem 重组糖蛋白产生的要求。 腺体已经演变为数百万年，如高效的丝绸蛋白质生产和分泌器官， 可以设计有效的产品并分泌重组蛋白 由于内源性蛋白蛋白 丝绸腺的糖基化途径无法正确地脱外糖基化的外源性真核糖蛋白。 拟议的研究试图开发蚕作为重组人的新型系统 通过创建编码需要一组需要的一组巨大酶的转基因蚕来产生糖蛋白 “人性化”天然丝绸蛋白N-糖基化途径和重组人N-糖蛋白 兴趣。 表达中间丝绸腺。 据我们所知，有一个蛋白质的有效遗传工程的一个先生 任何多细胞动物中的糖基化途径，包括B. Mori。 胶合制造蛋白质的蛋白质N-糖基化途径，以显着提高丝的使用 作为重组糖蛋白产生和分泌的生物反应器的腺体。 关于全球发达和欠发达国家的丝绸工业，允许增值产品 除基本丝绸外，对人类健康至关重要 纤维，从而大大提高了该外食生物制造平台的价值。 Jarvis和Fraser实验室具有执行这项研究的能力，如他们的 出版记录。 多年，产生大量相关的初步数据。 两个实验室，他们已建立的工作关系以及迄今为止获得的初步数据表明您 拟议的研究可以成功完成。