Integral membrane protein overexpression using organ bioreactors

描述（由申请方提供）：本研究项目的目的是生产足够数量的功能性重组哺乳动物整合膜蛋白（IMP），以用于结构生物学研究（X射线、二维电子晶体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，将有必要利用哺乳动物细胞的内在蛋白质折叠能力，以产生纯化的IMP需要详细研究其天然构象。膜蛋白包括一个非常大的比例-高达33% -的预测蛋白质的所有生物体检查到目前为止。它们在许多细胞和生理过程中起着至关重要的作用，例如作为细胞与其环境之间、细胞内隔室之间以及组成器官系统的隔室之间的物质和信息传递的重要介质。此外，膜蛋白对健康至关重要，并且已经是目前使用的绝大多数药物的目标。然而，对膜蛋白结构的理解，只代表了迄今为止解决的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经解决了蛋白质，可以从天然丰富的来源。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。本提案的具体目的是设计一种方法，以有效生产功能性、正确折叠的哺乳动物IMP，其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并行方法：1。具有生物素受体结构域（BAD）的重组蛋白的转基因表达既可以增加功能性IMP和多亚基IMP复合物的分离效率，足以使用“天然”器官表达，也可以用于在肌肉和肝脏中诱导“过表达”IMP作为体内器官生物反应器。2.具有生物素受体结构域（BAD）的重组IMP在肌肉和肝脏中作为Wt动物的体内器官生物反应器的直接急性表达可用于直接或诱导性地“过表达”IMP用于结构分析。作为原理的证明，我们将表达RyR 1，2.2MDa同源四聚体ER蛋白，445 Kda截短的C-末端RyR 1，和Cav1.1，5亚基骨骼肌慢电压门控Ca 2+通道。

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.