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理解生命所必需的基本相互作用是必不可少的。然而，获得用于基础研究和药物筛选的纯蛋白质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，特别是那些结合到细胞膜上的蛋白质。只有一小部分已知的蛋白质得到了彻底的表征。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，但它们只占已破译的3D蛋白质结构的0.5%。我们提出了一种系统，允许以可溶的、全功能的形式强大地生产和分离蛋白质。它将特别适合于膜蛋白的分析，并将适合于大规模筛选活性膜蛋白。我们计划利用新的克隆载体，这些载体可以稳定地保持原本“无法克隆”的基因。一种新的大肠杆菌细胞系将被开发出来，它可以在容易纯化的小泡中产生重组膜蛋白。该系统还将加入一种新的亲和捕捉剂，可产生95%纯度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。新型表达载体、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，否则这些蛋白将很难或不可能生产出来。这项技术将在生物技术中得到几乎普遍的应用，包括在制药研究(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。深入了解原核生物和真核生物中膜蛋白的结构和功能，可以为人类和动物的健康带来巨大的好处。例如，在激活状态下表达与药物相关的膜蛋白的能力，如离子通道和G蛋白偶联受体，可能会导致许多疾病的新疗法。由于细胞膜上多药外排蛋白的出现，许多癌症治疗方法变得无效，而对这些蛋白的更好理解可能会导致癌症治疗的改进。