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: 7658832
• 负责人: Paul D Allen
• 金额: $ 20.24万
• 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/7658832
• 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; 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; 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; overexpression; particle; prevent; programs; promoter; protein complex; protein folding; protein function; protein purification; protein structure; research study; scale up; 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（由申请人提供）：该研究项目的目的是产生功能性重组哺乳动物积分膜蛋白（IMP），足够数量，可用于结构生物学研究（X射线，2D电子晶体学和单个颗粒晶体冷冻电子显微镜）。我们的假设是，要实现这一目标，有必要利用哺乳动物细胞的固有蛋白质折叠能力来产生对其天然构型进行详细研究所需的纯化IMP。到目前为止，膜蛋白包含一个非常大的比例（最高33％）。它们在许多细胞和生理过程中起着至关重要的作用，例如充当细胞及其环境之间的材料和信息转移，细胞内部之间以及包括器官系统的隔室之间的重要介体。另外，膜蛋白对健康至关重要，已经是目前正在使用的绝大多数药物的目标。然而，对膜蛋白结构的理解仅表示迄今为止解决结构数量的一小部分。这样的结构信息不仅可以为膜蛋白功能的结构功能分析提供基础，而且可以促进药物发现的效率。因此，到目前为止，大多数膜蛋白结构已用于可以从天然富含来源获得的蛋白质。但是，许多人类生理和药物相关性最大的蛋白质具有相对较低的丰度。该提案的具体目的是设计一种方法，以有效地产生功能，适当折叠的哺乳动物imp，足以进行高分辨率的结构研究。 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。与生物素受体结构域（不良）在肌肉和肝脏中的直接急性表达一样，如体内器官生物反应器，可用于直接或直接或诱导的“过表达” IMPS进行结构分析。作为原理的证明，我们将表达RyR1，2.2MDA同型ER蛋白，445KDA截短的C末端RYR1和CAV1.1，5个亚基骨骼肌缓慢电压门控Ca2+通道。

项目成果

Three-dimensional localization of the α and β subunits and of the II-III loop in the skeletal muscle L-type Ca2+ channel.

骨骼肌 L 型 Ca2 通道中 α 和 β 亚基以及 II-III 环的三维定位。

• DOI: 10.1074/jbc.m112.419283
• 发表时间: 2012
• 期刊: The Journal of biological chemistry
• 作者: Szpyt,John;Lorenzon,Nancy;Perez,ClaudioF;Norris,Ethan;Allen,PaulD;Beam,KurtG;Samsó,Montserrat
• 通讯作者: Samsó,Montserrat