Overexpression of membrane proteins in Tetrahymena

描述（由申请人提供）：这些研究旨在建立四膜虫作为高通量结构研究的替代表达系统，并用于对人类健康重要的膜蛋白的大规模生产。为此，我们将尝试在该系统中使用一个独特的、多功能的高拷贝数克隆载体表达3种模型膜蛋白。该载体（目前正在构建中）利用了四膜虫的一个新的生物学特征，其中核糖体基因作为自主复制的回文小染色体的一部分被扩增到约18,000拷贝数。使用缺乏多克隆位点和亲和标签的类似载体，我们已经能够在四膜虫中表达外源gpi锚定的膜蛋白，其水平大于细胞总蛋白的3%。我们现在想在人类重要的跨膜蛋白上验证这种方法。这包括人类表皮生长因子受体（一种g蛋白偶联受体）和嗜离子性谷氨酸受体GluRG（作为通道蛋白）。四膜虫具有许多独特的特征，使其与更传统的蛋白质表达系统区别开来，并使其成为生产重组真核生物膜蛋白的理想载体。在这种情况下，一个成功的结果，功能蛋白的过表达，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使用的有吸引力的工具。我们的小企业合作伙伴，Tetragenetics公司，正在寻求将这项技术商业化，并通过蛋白质表达试剂盒和合同表达服务，将其提供给学术和工业领域的研究人员。真核生物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，最终导致从癌症到中风等严重疾病的治疗策略。

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.