HIGH THROUGHPUT CLONING OVEREXPRESSION AND PURIFICATION OF ACTIVE MEMBRANE PROT

活性膜保护的高通量克隆过表达和纯化

• 批准号: 7325336
• 负责人: DAVID Alan MEAD
• 金额: $ 25万
• 依托单位: LUCIGEN CORPORATION
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-02 至 2009-08-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7325336
• 关键词: Affinity; Affinity Chromatography; Animals; Basic Science; Binding; Binding Proteins; Biotechnology; Brain; Cell Line; Cell membrane; Cellular Membrane; Cloning; Cloning Vectors; Crystallography; Culture Media; Development; Disease; Escherichia coli; Eukaryota; Eukaryotic Cell; G Protein-Coupled Receptor Genes; G-Protein-Coupled Receptors; Generations; Genes; Genetic; Goals; Health; Human; Immunity; Individual; Inflammation; Intracellular Membranes; Ion Channel; Lactose; Lead; Life; Malignant Neoplasms; Membrane; Membrane Proteins; Microbial Biofilms; Modification; Organism; Pharmacologic Substance; Phase; Plasmids; Polymerase Chain Reaction; Preclinical Drug Evaluation; Production; Prokaryotic Cells; Protein Analysis; Protein Overexpression; Proteins; Reagent; Recombinants; Reporter; Research; Research Personnel; Screening procedure; Structure; System; Technology; Therapeutic; Vesicle; antimicrobial; cancer therapy; expression cloning; expression vector; improved; novel; protein expression; protein purification; protein structure; structural genomics

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.

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至关重要。然而，获得纯蛋白用于基础研究和药物筛选受到技术瓶颈的限制。例如，高通量研究受到生产和检测活性蛋白质的困难的严重阻碍，特别是那些与细胞膜结合的蛋白质。只有一小部分已知的蛋白质已被彻底表征。其中绝大多数是可溶性蛋白质。虽然膜蛋白可能构成生物体中基因的三分之一，但它们仅占已破译的3D蛋白质结构的0.5%。我们提出了一个系统，允许强大的生产和分离蛋白质的可溶性，功能齐全的形式。它将特别适合于膜蛋白的分析，并且它将适合于以大规模形式筛选活性膜蛋白。我们计划利用新的克隆载体，稳定地保持否则“不可克隆”的基因。一个新的E.大肠杆菌细胞系将开发生产重组膜蛋白在容易纯化的囊泡。该系统还将包含一种新的亲和捕获试剂，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质，否则将难以或不可能生产。这项技术将在生物技术中具有几乎普遍的应用，包括在药物研究（抗菌剂，免疫力，炎症）和环境研究（生物膜，微生物能源生产）中的关键重要性。对原核生物和真核生物中膜蛋白的结构和功能的更好理解可能对人类和动物健康产生重大益处。例如，在活性状态下表达药物相关膜蛋白（如离子通道和G蛋白偶联受体）的能力可能导致许多疾病的新疗法。由于细胞膜中多药外排蛋白的出现，许多癌症治疗变得无效，并且对这些蛋白质的更好理解可能导致改进的癌症治疗。