Overexpression of membrane proteins in Tetrahymena

四膜虫膜蛋白的过度表达

• 批准号: 7053444
• 负责人: THEODORE G CLARK
• 金额: $ 9.91万
• 依托单位: TETRAGENETICS, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-06 至 2007-03-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7053444
• 关键词: Tetrahymena; biotechnology; expression cloning; gene expression; high throughput technology; membrane proteins; natural gene amplification; protein purification; protein structure; protozoal genetics; recombinant proteins; ribosomes; structural biology; technology /technique development; transfection /expression vector

DESCRIPTION (provided by applicant): These studies are intended to establish Tetrahymena as an alternative expression system for use in high-throughput structural studies, and for large-scale production of membrane proteins with importance in human health. Towards that end, we will attempt to express 3 model membrane proteins in this system using a unique and versatile high-copy number cloning vector. The vector (now under construction) takes advantage of a novel biological feature of Tetrahymena in which the ribosomal genes are amplified to ~ 18,000 copy number as part of an autonomously replicating palindromic minichromosome. Using a similar vector that lacks a multicloning site and affinity tags, we have been able to express a heterologous GPI-anchored membrane protein in Tetrahymena to levels greater than 3% of total cell protein. We now want to validate this approach for important transmembrane proteins in humans. This includes the human epidermal growth factor receptor (a G-protein coupled receptor) and the ionotropic glutamate receptor GluRG (which acts as a channel protein). Tetrahymena has a number of distinct features which set it apart from more conventional protein expression systems and which make it ideal as a vehicle for the production of recombinant eucaryotic membrane proteins. A successful outcome, in this case overexpression of functional proteins, will provide proof-of-principal for the use of this system and make it an attractive tool for use by the biopharmaceutical industry. Our Small Business Partner, Tetragenetics Inc, seeks to commercialize on this technology and make it available to researchers in both the academic and industry arenas through protein expression kits and contract expression services. Eucaryotic membrane proteins contribute to an array of vital cell functions and are essential to human health. The technology described here is intended to better understand how these proteins function, leading ultimately to strategies for the treatment of serious disorders ranging from cancer to stoke.

描述（由申请人提供）：这些研究旨在建立四膜虫作为替代表达系统，用于高通量结构研究以及对人类健康具有重要意义的膜蛋白的大规模生产。为此，我们将尝试使用独特且通用的高拷贝数克隆载体在该系统中表达 3 种模型膜蛋白。该载体（目前正在构建）利用了四膜虫的一种新生物学特征，其中核糖体基因被扩增至约 18,000 个拷贝数，作为自主复制回文微型染色体的一部分。使用缺乏多克隆位点和亲和标签的类似载体，我们能够在四膜虫中表达异源 GPI 锚定膜蛋白，其水平高于总细胞蛋白的 3%。我们现在想要验证这种方法对人类重要的跨膜蛋白的影响。这包括人表皮生长因子受体（G 蛋白偶联受体）和离子型谷氨酸受体 GluRG（充当通道蛋白）。四膜虫具有许多独特的特征，使其有别于更传统的蛋白质表达系统，并使其成为生产重组真核膜蛋白的理想载体。成功的结果（在本例中是功能蛋白的过度表达）将为该系统的使用提供原理证明，并使其成为生物制药行业有吸引力的工具。我们的小型企业合作伙伴 Tetra Genetics Inc 寻求将该技术商业化，并通过蛋白质表达试剂盒和合约表达服务向学术和工业领域的研究人员提供该技术。真核膜蛋白有助于一系列重要的细胞功能，对人类健康至关重要。这里描述的技术旨在更好地了解这些蛋白质的功能，最终制定治疗从癌症到中风等严重疾病的策略。