HIGH THROUGHPUT CLONING OVEREXPRESSION AND PURIFICATION OF ACTIVE MEMBRANE PROT

描述（由申请人提供）：蛋白质的功能分析对于彻底了解生命所必需的基本相互作用是必不可少的。然而，获得纯蛋白质用于基础研究和药物筛选受到技术瓶颈的限制。例如，由于难以产生和检测活性蛋白，特别是那些结合在细胞膜上的活性蛋白，高通量研究受到严重阻碍。只有一小部分已知的蛋白质被彻底地表征了。其中绝大多数是可溶性蛋白质。尽管膜蛋白可能构成了生物体中三分之一的基因，但它们只代表了已被破译的三维蛋白质结构的0.5%。我们提出了一个系统，允许稳健的生产和分离蛋白质在可溶性，全功能的形式。它将特别适合于膜蛋白的分析，并且它将适用于大规模筛选活性膜蛋白。我们计划利用新的克隆载体，稳定地维持否则“不可克隆”的基因。一种新的大肠杆菌细胞系将在易于纯化的囊泡中产生重组膜蛋白。该系统还将纳入一种新的亲和力捕获试剂，可产生纯度为95%的活性未变性蛋白质。在第二阶段，我们计划扩展这项技术，以促进最困难的蛋白质的表达。新的表达载体、细胞系和纯化试剂将被开发成单独的产品和完整的试剂盒。这些产品将使研究人员能够研究各种各样的可溶性或膜结合蛋白，否则将很难或不可能生产。这项技术将在生物技术中几乎普遍应用，包括在药物研究（抗菌剂、免疫、炎症）和环境研究（生物膜、微生物能源生产）中至关重要。对原核生物和真核生物膜蛋白结构和功能的进一步了解将对人类和动物的健康产生重大的益处。例如，在活性状态下表达与药物相关的膜蛋白（如离子通道和G蛋白偶联受体）的能力可能会导致许多疾病的新疗法。由于细胞膜中出现了多药物外排蛋白，许多癌症治疗方法变得无效，对这些蛋白的进一步了解可以改善癌症治疗。

DESCRIPTION (provided by applicant): Functional analysis of proteins is essential to thoroughly understand the basic interactions essential for life. However, obtaining pure proteins for basic research and drug screening is limited by technical bottlenecks. For example, high throughput studies are severely hampered by difficulties in producing and detecting active proteins, especially those that are bound to cellular membranes. Only a small fraction of known proteins have been thoroughly characterized. Of these the vast majority are soluble proteins. Although membrane proteins may constitute a third of the genes in an organism, they represent only 0.5% of the 3D protein structures that have been deciphered. We propose a system to allow robust production and isolation of proteins in a soluble, fully functional form. It will be particularly well-suited to analysis of membrane proteins, and it will be amenable to screening for active membrane proteins in a large-scale format. We plan to make use of novel cloning vectors that stably maintain otherwise "unclonable" genes. A new E. coli cell line will be developed that produces recombinant membrane proteins in easily-purified vesicles. This system will also incorporate a new affinity capture reagent that yields active, undenatured protein with 95% purity. In Phase II, we plan to extend this technology to facilitate expression of the most difficult proteins. Novel expression vectors, cell lines, and purification reagents will be developed into individual products and complete kits. These products will enable researchers to study a wide variety of soluble or membrane- bound proteins that otherwise would be difficult or impossible to produce. This technology will have nearly universal applications in biotechnology, including critical importance in pharmaceutical research (antimicrobials, immunity, inflammation) and environmental research (biofilms, microbial energy production). An improved understanding of the structure and function of membrane proteins in prokaryotes and eukaryotes could result in significant benefits for human and animal health. For example, the ability to express pharmaceutically relevant membrane proteins, such as ion channels and G protein-coupled receptors, in an active state could lead to new therapies for numerous diseases. Many cancer therapeutics become ineffective due to the emergence of multidrug efflux proteins in the cell membrane, and an improved understanding of these proteins could result in improved cancer therapies.