Integral membrane protein overexpression using organ bioreactors

使用器官生物反应器过度表达整合膜蛋白

• 批准号: 7313034
• 负责人: Paul D Allen
• 金额: $ 20.09万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-15 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7313034
• 关键词: Acute; Animal Organ; Animals; Basement membrane; Biomass; Bioreactors; Biotin; Brain; C-terminal; Cell Culture System; Cells; Complementary DNA; Complex; Cryoelectron Microscopy; Disadvantaged; Elements; Environment; Future; Goals; HSV vector; Health; Human; In Situ; In Vitro; Integral Membrane Protein; Lifting; Liver; Mammalian Cell; Mediator of activation protein; Membrane Proteins; Methods; Modeling; Molecular Conformation; Mus; Muscle; N-terminal; Numbers; Operative Surgical Procedures; Organ; Organ Harvestings; Organism; Peripheral; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Physiological; Physiological Processes; Pituitary Gland; Play; Portal vein structure; Principal Investigator; Production; Protein Overexpression; Protein Subunits; Proteins; Purpose; Rattus; Recombinant Proteins; Recombinants; Research Personnel; Research Project Grants; Resolution; Retina; Roentgen Rays; Role; RyR1; Serotyping; Simplexvirus; Skeletal Muscle; Solutions; Source; Streptavidin; Structure; System; Testing; Thick; Time; Tissues; Transgenes; Transgenic Animals; Transgenic Organisms; Veins; Viral; Virion; Virus; base; body system; drug discovery; electron crystallography; experience; human disease; in vivo; novel; particle; prevent; programs; promoter; protein folding; protein function; protein purification; protein structure; research study; scale up; size; small molecule; structural biology; voltage

DESCRIPTION (provided by applicant): The object of this research project is to produce functional recombinant mammalian integral membrane proteins (IMPs) in sufficient quantities to be useful for structural biology studies (X-ray, 2D electron crystallography and Single Particle Cryo-Electron microscopy). Our hypothesis is that to accomplish this goal it will be necessary to utilize the intrinsic protein folding capacity of mammalian cells to produce the purified IMPs needed for a detailed study of their native conformation. Membrane proteins comprise a very substantial proportion - up to 33% - of the predicted proteins of all organisms examined so far. They play a crucial role in many cellular and physiological processes such as acting as essential mediators of material and information transfer between cells and their environment, between compartments within cells, and between compartments comprising organ systems. In addition membrane proteins are vital to health and are already the target of the vast majority of drugs currently in use. An understanding of the structure of membrane proteins, however, represents only tiny fraction of the number of solved structures to date. Such structural information could significantly not only provide a basis for structure function analysis of membrane protein function but could facilitate the efficiency of drug discovery. Thus so far, most membrane protein structures have been solved for proteins that can be obtained from naturally rich sources. However, many of the proteins of greatest human physiological and pharmaceutical relevance are of relatively low abundance. The SPECIFIC AIM of this proposal is to devise a method to efficiently produce functional, properly folded mammalian IMPs in quantities sufficient to conduct high-resolution structural studies. To accomplish this aim we will test two novel parallel approaches: 1. Transgenic expression of a recombinant protein with a biotin acceptor domain (BAD) can both increase the efficiency of isolation of functional IMPs and multiple subunit IMP complexes sufficiently to use "native" organ expression or can be used to inducibly "over-express" IMPs in muscle and liver as in vivo organ bioreactors. 2. Direct acute expression of a recombinant IMP with a biotin acceptor domain (BAD) in muscle and liver as in vivo organ bioreactors of Wt animals can be used to directly or inducibly "over-express" IMPs for structural analysis. As proof of principle, we will express RyR1, a 2.2MDa homotetrameric ER protein, a 445Kda truncated C-terminal RyR1, and Cav1.1, the 5 subunit skeletal muscle slow voltage gated Ca2+channel.

描述(申请人提供)：本研究项目的目标是生产足够数量的功能性重组哺乳动物整膜蛋白(IMPS)，用于结构生物学研究(X射线、2D电子结晶学和单粒子冷冻电子显微镜)。我们的假设是，为了实现这一目标，有必要利用哺乳动物细胞固有的蛋白质折叠能力来产生纯化的IMP，这是详细研究其天然构象所需的。膜蛋白在到目前为止研究过的所有生物体的预测蛋白中占很大比例--高达33%。它们在许多细胞和生理过程中发挥着至关重要的作用，如作为细胞与其环境之间、细胞内隔间和组成器官系统的隔间之间物质和信息传递的基本媒介。此外，膜蛋白对健康至关重要，并且已经成为目前使用的绝大多数药物的靶标。然而，对膜蛋白结构的了解只占迄今为止已解决结构数量的一小部分。这些结构信息不仅可以为膜蛋白功能的结构功能分析提供重要的依据，而且有助于药物发现的效率。到目前为止，大多数膜蛋白质结构已经被解决，可以从天然丰富的来源获得蛋白质。然而，许多与人类生理和药物相关性最大的蛋白质丰度相对较低。这一建议的具体目的是设计一种方法，高效地生产具有功能、适当折叠的哺乳动物IMP，数量足以进行高分辨率结构研究。为了实现这一目标，我们将测试两种新的平行方法：1.转基因表达带有生物素受体结构域(BAD)的重组蛋白既可以提高分离功能IMP和多亚单位IMP复合体的效率，充分利用“天然”器官表达，也可以用来在肌肉和肝脏诱导“过表达”IMP作为体内器官生物反应器。2.将含有生物素受体结构域(BAD)的重组IMP在肌肉和肝脏中直接急性表达，作为Wt动物体内器官生物反应器，可以直接或诱导地“过表达”IMP，用于结构分析。作为原理的证明，我们将表达RyR1，一个2.2MDa的同源四聚体ER蛋白，一个445Kda截短的C端RyR1，以及Cav1.1，一个5亚单位的骨骼肌慢电压门控钙通道。