一种用于重组糖蛋白生产的新型转基因家蚕系统

Abstract

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.

摘要：

许多生物医学上重要的蛋白质，包括抗体，细胞因子，抗凝血剂，血液凝固因子等，都是糖蛋白。因此，对可用于生产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质生产系统不能满足这种需求。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产生大量的重组糖蛋白。该提议的长期目标是对家蚕进行基因工程以满足这些要求，并为重组糖蛋白的生产提供新的系统。最近的研究表明，家蚕丝腺是一种高效的丝蛋白生产和分泌器官，可以进行基因工程改造，以有效地生产和分泌重组蛋白。但是，转基因蚕既没有开发也没有用于重组糖蛋白的生产。主要障碍是不能期望丝腺的内源蛋白质糖基化途径适当地糖基化高等真核糖蛋白。我们将使用代谢工程来克服这一障碍，作为开发家蚕作为重组人糖蛋白生产新系统的更广泛努力的一部分。基本方法是使用piggyBac载体系统分离转基因蚕，其编码（1）人源化天然丝腺蛋白N-糖基化途径所需的高等真核酶和（2）重组人N-糖蛋白。每个转基因将置于组织特异性启动子的控制之下，该启动子将其表达靶向丝腺。以前没有关于使用任何类型的转基因昆虫作为生物反应器生产重组糖蛋白的报道。此外，以前没有关于在任何多细胞动物中设计蛋白质糖基化途径的报道。因此，使用转基因家蚕的丝腺作为重组糖蛋白生成和分泌的生物反应器的提议，以及在该低等真核生物的主要器官中代谢工程化蛋白质N-糖基化途径的提议，是真正的原创和创新。该提议的具体目的是（1）构建和测试piggyBac载体，用于丝腺特异性表达编码（1a）人源化家蚕蛋白N-糖基化途径所需的酶的基因和（1b）编码重组人糖蛋白的基因。 （2）使用来自目标1的piggyBac载体产生转基因蚕;和（3）评估来自目标2的转基因蚕的重组糖蛋白产生，分泌和糖基化。糖蛋白是蛋白质的主要亚类。存在与多肽骨架共价连接的碳水化合物侧链。许多不同类型的生物医学上重要的蛋白质，例如抗体，细胞因子，抗凝血剂，凝血因子是糖蛋白。研究人类糖蛋白或将其用于临床用途的现代生物医学研究人员严重依赖于重组蛋白质生产系统。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于重组糖蛋白的生产，因为很少有人可以产生具有真正碳水化合物侧链的高等真核糖蛋白。因此，本文提出的研究的基本目的是创建可用于生产用于基础生物医学研究和直接临床应用的重组糖蛋白的新系统。更具体地说，我们将通过遗传工程改造家蚕来创造这个新系统。虽然用蚕等毛虫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。 一个主要原因是蚕丝腺作为高效蛋白质生产和分泌器官已经进化了数百万年。此外，一些已发表的研究表明，该器官可以被设计为有效地产生和分泌重组蛋白。然而，蚕未用于重组糖蛋白生产，因为它们的内源蛋白糖基化途径不能正确地糖基化外来的高等真核糖蛋白。 Jarvis和Fraser实验室共同致力于解决这一问题。 Jarvis实验室在过去十年中一直在研究和设计昆虫蛋白质糖基化途径，并且Fraser实验室已经开发出一种用于昆虫特别是家蚕的有效遗传转化的卓越系统。因此，我们计划结合我们的技能来分离转基因蚕，其将编码人源化其蛋白质糖基化途径所需的高等真核酶和感兴趣的生物医学相关的人糖蛋白。重要的是，每种转基因的表达将特异性地靶向使用组织特异性启动子放置的丝腺。以前没有关于使用任何类型的转基因昆虫作为生物反应器生产重组糖蛋白的报道。以前也没有关于在任何多细胞动物中设计蛋白质糖基化途径的报道。因此，我们建议使用转基因家蚕的丝腺作为重组糖蛋白生成和分泌的生物反应器，再加上我们在这种低等真核生物的主要器官中代谢工程化蛋白质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影响。更好的重组糖蛋白生产工具将促进糖蛋白结构和功能的基础研究。它还可以用于生物技术工业中以生产用于临床用作疫苗或治疗剂的重组糖蛋白。同样，虽然它看起来像一个奇怪的平台，但使用毛虫生产非糖基化蛋白质的想法已经商业化（见www.c-perl.com）。考虑到许多高知名度，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“）是糖蛋白，该系统的生物技术影响可能很大。代谢工程的努力，这是该项目的关键，代表了一个精心设计的异位表达实验，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。这些结果将引起人们极大的兴趣。科学家，特别是研究低等生物中蛋白质N-糖基化的糖生物学家以及蛋白质糖基化途径的进化。最后，这些结果将对生物工程师非常感兴趣，他们正在努力克服低等真核生物系统对重组糖蛋白生成的进化限制。

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