A novel transgenic silkworm system for recombinant glycoprotein production

一种用于重组糖蛋白生产的新型转基因蚕系统

• 批准号: 7368649
• 负责人: Donald L. Jarvis
• 金额: $ 29.93万
• 依托单位: UNIVERSITY OF WYOMING
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-29 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7368649
• 关键词: Address; Animals; Antibodies; Anticoagulants; Area; Basic Science; Biological; Biomedical Engineering; Biomedical Research; Bioreactors; Biotechnology; Blood Clot; Blood Coagulation Factor; Blood coagulation; Bombyx; Carbohydrates; Cell Line; Cells; Clinical; Coupled; Data; Development; Ectopic Expression; Engineering; Enzymes; Epoetin Alfa; Eukaryota; Eukaryotic Cell; Evolution; Gene Expression; Gene Transfer; Genes; Genetic Transformation; Gland; Glycoproteins; Human; Industry; Insect Proteins; Insecta; Link; Lower Organism; Metabolic; Methods; Organ; Pathway interactions; Positioning Attribute; Process; Production; Protein Engineering; Protein Glycosylation; Proteins; Publishing; Purpose; Recombinant Proteins; Recombinants; Records; Reporting; Research; Research Personnel; Roche brand of trastuzumab; Scientist; Side; Silk; System; Tenecteplase; Testing; Therapeutic; Tissues; Today; Transgenes; Transgenic Organisms; Vaccines; Vertebral column; Work; clinical application; clinically relevant; cost; cytokine; experience; glycoprotein structure; glycosylation; innovation; interest; novel; polypeptide; promoter; research study; skills; tool; vector

DESCRIPTION (provided by applicant): 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. However, currently available recombinant protein production systems cannot meet this demand. In fact, no currently available system can produce large amounts of recombinant glycoproteins in properly glycosylated form at relatively low cost. The long-term objective of this proposal is to genetically engineer the silkworm to fulfill these requirements and provide a new system for recombinant glycoprotein production. Recent studies have shown that the silkworm silk gland, which is a highly efficient silk protein production and secretion organ, can be genetically engineered to efficiently produce and secrete recombinant proteins. But, transgenic silkworms have been neither developed nor used for recombinant glycoprotein production. The major impediment is that the endogenous protein glycosylation pathways of the silk gland cannot be expected to properly glycosylate higher eukaryotic glycoproteins. We will use metabolic engineering to overcome this impediment, as part of a broader effort to develop the silkworm as a new system for recombinant human glycoprotein production. The basic approach will be to use the piggyBac vector system to isolate transgenic silkworms encoding (1) higher eukaryotic enzymes needed to humanize the native silk gland protein N-glycosylation pathway and (2) a recombinant human N-glycoprotein. Each transgene will be placed under the control of a tissue-specific promoter that will target its expression to the silk gland. There are no previous reports of recombinant glycoprotein production using any type of transgenic insect as a bioreactor. In addition, there are no previous reports of engineering a protein glycosylation pathway in any multicellular animal. Therefore, the proposal to use the silk gland of a transgenic silkworm as a bioreactor for recombinant glycoprotein production and secretion, coupled with the proposal to metabolically engineer the protein N-glycosylation pathway in a major organ of this lower eukaryote, is truly original and innovative. The specific aims of this proposal are (1) To construct and test piggyBac vectors for silk gland-specific expression of genes encoding (1a) enzymes needed to humanize the silkworm protein N-glycosylation pathway and (1b) genes encoding a recombinant human glycoprotein of interest; (2) To use the piggyBac vectors from aim 1 to produce transgenic silkworms; and (3) To assess recombinant glycoprotein production, secretion, and glycosylation by the transgenic silkworms from aim 2.The glycoproteins are a major subclass of proteins distinguished by the presence of carbohydrate side chains covalently linked to the polypeptide backbone. Many different types of biomedically significant proteins, such as antibodies, cytokines, anticoagulants, blood clotting factors are glycoproteins. Modern biomedical researchers studying human glycoproteins or producing them for clinical use rely heavily on recombinant protein production systems. Thus, there is a high demand for systems that can be used to produce recombinant glycoproteins. Unfortunately, few of the currently available systems are well suited for the production of recombinant glycoproteins, as few can produce higher eukaryotic glycoproteins with authentic carbohydrate side chains. Thus, the basic purpose of the research proposed herein is to create a new system that can be used to produce recombinant glycoproteins for basic biomedical research and direct clinical applications. More specifically, we will genetically engineer the silkworm to create this new system. While it might seem strange to target a caterpillar, such as the silkworm, to develop a recombinant glycoprotein production system, we have good reasons to do so. One major reason is that the silkworm silk gland has evolved over millions of years as a highly efficient protein production and secretion organ. Furthermore, several published studies have shown that this organ can be engineered to efficiently produce and secrete recombinant proteins. However, silkworms have not been used for recombinant glycoprotein production because their endogenous protein glycosylation pathways cannot properly glycosylate foreign, higher eukaryotic glycoproteins. Together, the Jarvis and Fraser labs are uniquely positioned to address this problem. The Jarvis lab has been studying and engineering insect protein glycosylation pathways for the past decade and the Fraser lab has developed a superb system for efficient genetic transformation of insects, particularly the silkworm. Thus, we plan to combine our skills to isolate transgenic silkworms that will encode both the higher eukaryotic enzymes needed to humanize their protein glycosylation pathway and a biomedically relevant human glycoprotein of interest. Importantly, the expression of each transgene will be specifically targeted to the silk gland placed using a tissue-specific promoter. There are no previous reports of recombinant glycoprotein production using any type of transgenic insect as a bioreactor. There also are no previous reports of engineering a protein glycosylation pathway in any multicellular animal. Therefore, our proposal to use the silk gland of a transgenic silkworm as a bioreactor for recombinant glycoprotein production and secretion, coupled with our proposal to metabolically engineer the protein N-glycosylation pathway in a major organ of this lower eukaryote, is truly original and innovative. The successful development of the silkworm as a system for recombinant glycoprotein production would have a broad impact with implications in many areas of biomedical research. A better tool for recombinant glycoprotein production would facilitate basic research on glycoprotein structure and function. It also could be used in the biotechnology industry to produce recombinant glycoproteins for clinical use as vaccines or therapeutics. Again, while it might seem like a strange platform, the idea to use caterpillars for the production of non-glycosylated proteins has already been commercialized (see www.c-perl.com). The biotechnological impact of this system could be huge, considering that many high profile, clinically relevant proteins, such as antibodies (e.g. herceptin.), cytokines (e.g. EPOGEN), and anticoagulants (e.g. Tenecteplase") are glycoproteins. At a more basic level, the metabolic engineering effort, which is key to this project, represents an elaborate ectopic expression experiment that will broadly address the biological significance of the differences in protein N-glycosylation pathways of lower and higher eukaryotes. These results will be of great interest to basic scientists, particularly glycobiologists studying protein N-glycosylation in lower organisms and the evolution of protein glycosylation pathways. Finally, these results will be of great interest to bioengineers working to overcome the evolutionary limitations of lower eukaryotic systems for recombinant glycoprotein production.

描述（由申请人提供）：许多具有生物医学显着的蛋白质，包括抗体，细胞因子，抗凝剂，血液凝血因子等，都是糖蛋白。因此，对系统的需求很高，可用于生产重组糖蛋白，用于基本的生物医学研究和直接临床应用。但是，目前可用的重组蛋白质生产系统无法满足这一需求。实际上，目前没有可用的系统可以以相对较低的成本以适当的糖基化形式产生大量的重组糖蛋白。该提案的长期目标是遗传设计蚕，以满足这些要求，并为重组糖蛋白生产提供新的系统。最近的研究表明，蚕丝腺是一种高效的丝绸蛋白质生产和分泌器官，可以在遗传上设计，以有效地产生和分泌重组蛋白。但是，转基因蚕未开发，也不用于重组糖蛋白的产生。主要障碍是，不能期望丝腺的内源性蛋白质糖基化途径正确地糖基化较高的真核糖蛋白。我们将使用代谢工程来克服这一障碍，这是开发蚕的更广泛努力的一部分，作为重组人糖蛋白生产的新系统。基本方法是使用PiggyBac载体系统分离编码的转基因蚕（1）（1）将天然丝绸蛋白N-糖基化途径人性化所需的较高的真核酶，以及（2）重组人类N-糖蛋白。每个转基因将置于组织特异性启动子的控制下，该启动子将其表达靶向丝绸腺体。以前没有任何使用任何类型的转基因昆虫作为生物反应器的重组糖蛋白产生的报道。此外，以前没有关于任何多细胞动物中蛋白质糖基化途径进行工程的报道。因此，使用转基因蚕的丝腺作为重组糖蛋白产生和分泌的生物反应器的提议，再加上该蛋白质N-糖基化途径在该较低真主剂的主要器官中的代谢提案是真正的原始和创新的。该提案的具体目的是（1）构建和测试Piggybac向量，以用于编码基因（1A）酶的丝腺特异性表达（1A）酶，以人性化蚕蛋白N-糖基化途径和（1B）基因编码具有利益的重组人糖蛋白； （2）使用AIM 1的Piggybac向量产生转基因蚕； （3）评估来自AIM 2的转基因蚕的重组糖蛋白的产生，分泌和糖基化。糖蛋白是通过与多肽链链共链链接的碳水化合物侧链区分的蛋白质的主要子类。许多不同类型的生物医学有意义的蛋白质，例如抗体，细胞因子，抗凝剂，血液凝结因子是糖蛋白。研究人糖蛋白或生产临床用途的现代生物医学研究人员在很大程度上取决于重组蛋白质生产系统。因此，对可用于产生重组糖蛋白的系统的需求很高。不幸的是，当前可用的系统中很少有非常适合生产重组糖蛋白，因为很少有能够产生具有正宗碳水化合物侧链的较高真核糖蛋白。因此，本文提出的研究的基本目的是创建一个新系统，该系统可用于生产重组糖蛋白，用于基本的生物医学研究和直接临床应用。更具体地说，我们将在基因上设计蚕，以创建这个新系统。虽然针对毛毛虫（例如蚕），开发重组糖蛋白生产系统似乎很奇怪，但我们有充分的理由这样做。一个主要原因是，作为高效的蛋白质生产和分泌器官，蚕丝丝腺已经发展了数百万年。此外，几项已发表的研究表明，该器官可以设计为有效产生和分泌重组蛋白。然而，蚕未用于重组糖蛋白的产生，因为它们的内源性蛋白质糖基化途径无法正确地糖基化糖基化外溶外溶性，较高的真核糖蛋白。 Jarvis和Fraser Labs共同解决了这一问题。贾维斯实验室（Jarvis Lab）在过去十年中一直在研究和工程昆虫蛋白糖基化途径，而弗雷泽（Fraser）实验室开发了一种出色的系统，用于有效地遗传昆虫的遗传转化，尤其是蚕。因此，我们计划结合我们的技能，以分离转基因蚕，这将编码人性化其蛋白质糖基化途径所需的较高的真核酶和感兴趣的生物医学相关的人类糖蛋白。重要的是，每个转基因的表达将被特异性地针对使用组织特异性启动子放置的丝腺。以前没有任何使用任何类型的转基因昆虫作为生物反应器的重组糖蛋白产生的报道。以前还没有关于任何多细胞动物中蛋白质糖基化途径进行工程的报道。因此，我们提出的提议将转基因蚕的丝质腺作为重组糖蛋白产生和分泌的生物反应器，再加上我们在该较低真核生物的主要机构中代谢设计蛋白质N-糖基化途径的提议是真正的原始和创新的。作为重组糖蛋白产生的系统的成功发展，将在许多生物医学研究领域产生广泛的影响。重组糖蛋白产生的更好工具将促进有关糖蛋白结构和功能的基础研究。它也可以在生物技术行业中用于生产重组糖蛋白作为疫苗或治疗剂的临床用途。同样，虽然这似乎是一个奇怪的平台，但使用毛毛虫来生产非糖基化蛋白质的想法已经被商业化（请参阅www.c-perl.com）。考虑到许多抗体（例如赫赛替丁），细胞因子（例如，雌激素）和抗凝剂（例如，tenecteplase'glycopotins of glycopotins of基本水平，实验，它是代表的，它是代理的，这是一个促成，这是一个蛋白质，考虑到了，该系统是一个exportions，这是一个促成，因此，该系统的生物技术影响可能会很大广泛地解决了较低和较高的真核生物的蛋白质N-糖基化途径的生物学意义。用于重组糖蛋白的产生。