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: 7908796
• 负责人: Donald L. Jarvis
• 金额: $ 27.73万
• 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 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7908796
• 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; 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; Recombinant Proteins; Recombinants; Records; Reporting; Research; Research Personnel; Roche brand of trastuzumab; Scientist; Side; Silk; System; Tenecteplase; Testing; Therapeutic; Tissues; Transgenes; Transgenic Organisms; Vaccines; Vertebral column; Work; clinical application; clinically relevant; cost; cytokine; experience; glycoprotein structure; glycosylation; innovation; interest; meetings; 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）：许多具有生物医学意义的蛋白质，包括抗体、细胞因子、抗凝血剂、凝血因子等，都是糖蛋白。因此，对用于基础生物医学研究和直接临床应用的重组糖蛋白的系统有很高的需求。然而，目前可用的重组蛋白生产系统无法满足这一需求。事实上，目前没有可用的系统能够以相对低的成本以适当的糖基化形式生产大量重组糖蛋白。本提案的长期目标是对蚕进行基因工程改造，以满足这些要求，并提供重组糖蛋白生产的新系统。近年来的研究表明，家蚕丝腺是一种高效的丝蛋白生产和分泌器官，可以通过基因工程高效地生产和分泌重组蛋白。但是，转基因蚕既没有被开发出来，也没有被用于重组糖蛋白的生产。主要的障碍是丝腺的内源性蛋白糖基化途径不能期望正确地糖基化更高的真核糖蛋白。我们将利用代谢工程来克服这一障碍，作为开发蚕作为重组人糖蛋白生产新系统的更广泛努力的一部分。基本方法将是使用piggyBac载体系统分离编码：(1)人源化天然蚕丝腺蛋白n -糖基化途径所需的高级真核酶和(2)重组人n -糖蛋白的转基因蚕。每个转基因都将被置于组织特异性启动子的控制下，该启动子将针对丝腺的表达。利用任何类型的转基因昆虫作为生物反应器来生产重组糖蛋白，目前尚无报道。此外，在任何多细胞动物中，也没有工程蛋白糖基化途径的报道。因此，利用转基因蚕的蚕丝腺作为重组糖蛋白生产和分泌的生物反应器，再加上在这种低等真核生物的主要器官中代谢工程蛋白n -糖基化途径的提议，是真正的原创和创新。本提案的具体目的是：(1)构建和测试piggyBac载体，用于蚕丝腺特异性表达编码（1a）人源化家蚕蛋白n -糖基化途径所需酶的基因和（1b）编码重组人糖蛋白的基因；(2)利用目标1中的piggyBac载体培育转基因家蚕；(3)评价目标2转基因家蚕重组糖蛋白的产生、分泌和糖基化。糖蛋白是一种主要的蛋白质亚类，其特征是与多肽主链共价连接的碳水化合物侧链。许多不同类型的具有生物医学意义的蛋白质，如抗体、细胞因子、抗凝血剂、凝血因子等都是糖蛋白。现代生物医学研究人员研究人类糖蛋白或生产用于临床的糖蛋白在很大程度上依赖于重组蛋白生产系统。因此，对可用于生产重组糖蛋白的系统有很高的需求。不幸的是，目前可用的系统很少适合生产重组糖蛋白，因为很少有系统可以生产具有真正碳水化合物侧链的高级真核糖蛋白。因此，本文提出的研究的基本目的是创建一个新的系统，可用于生产重组糖蛋白的基础生物医学研究和直接临床应用。更具体地说，我们将对蚕进行基因工程改造，以创建这个新系统。虽然以毛毛虫为目标，比如蚕，来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。一个主要原因是蚕的丝腺已经进化了数百万年，成为一种高效的蛋白质生产和分泌器官。此外，一些已发表的研究表明，该器官可以被设计成有效地产生和分泌重组蛋白。然而，蚕还没有被用于重组糖蛋白的生产，因为它们的内源性蛋白糖基化途径不能正确地糖基化外来的、高级的真核糖蛋白。贾维斯和弗雷泽实验室在解决这一问题上具有独特的优势。贾维斯实验室在过去的十年里一直在研究和设计昆虫蛋白质糖基化途径，弗雷泽实验室已经开发出一种极好的系统，可以有效地对昆虫，特别是蚕进行遗传转化。因此，我们计划结合我们的技能分离转基因蚕，这些转基因蚕将编码人源化其蛋白糖基化途径所需的高级真核酶和生物医学上相关的人糖蛋白。重要的是，每个转基因的表达都将使用组织特异性启动子专门针对丝腺。利用任何类型的转基因昆虫作为生物反应器来生产重组糖蛋白，目前尚无报道。在任何多细胞动物中也没有工程蛋白糖基化途径的报道。因此，我们提出的利用转基因蚕丝腺作为重组糖蛋白生产和分泌的生物反应器，再加上我们提出的在这种低等真核生物的主要器官中代谢工程蛋白n -糖基化途径的建议，是真正的原创和创新。家蚕作为重组糖蛋白生产系统的成功开发将对生物医学研究的许多领域产生广泛的影响。一个更好的重组糖蛋白生产工具将有助于糖蛋白结构和功能的基础研究。它还可用于生物技术工业，生产用于临床的重组糖蛋白，如疫苗或治疗药物。再一次，虽然这看起来像是一个奇怪的平台，但利用毛毛虫生产非糖基化蛋白质的想法已经商业化了（见www.c-perl.com）。考虑到许多引人注目的临床相关蛋白，如抗体（如赫赛汀）、细胞因子（如EPOGEN）和抗凝血剂（如Tenecteplase）都是糖蛋白，该系统的生物技术影响可能是巨大的。在更基本的水平上，代谢工程的努力是这个项目的关键，它代表了一个复杂的异位表达实验，将广泛地解决低等和高等真核生物蛋白质n -糖基化途径差异的生物学意义。这些结果将引起基础科学家的极大兴趣，特别是糖生物学家在低等生物中研究蛋白质n -糖基化和蛋白质糖基化途径的进化。最后，这些结果将对致力于克服重组糖蛋白生产的低级真核系统的进化限制的生物工程师产生极大的兴趣。