Mammalian skeletal muscle: a recombinant protein factory

描述（由申请人提供）：本R21探索性研究资助响应NIGMS对开发新型真核表达系统的要求，该系统能够产生足够数量的纯膜蛋白，以进行结晶试验。这种系统的缺乏严重限制了整体膜蛋白结构研究的数量，如离子通道和转运体，具有原子水平的分辨率。目前的应用旨在通过研究哺乳动物骨骼肌在体内转染基因工程DNA质粒时产生大量重组膜蛋白的潜在用途来解决这一问题。我们认为，骨骼肌可能不仅是大量合成胞质蛋白的理想制剂，而且最重要的是，它可以生产和输出大量的跨膜蛋白。我们计划在两个独立但内在联系的特定目标中通过实验来追求这些想法。特异性目的1将测试用用于哺乳动物表达6 -组氨酸（6His）标记的EGFP和ECFP的piasmids在肌肉组织中的体内电穿孔是否允许我们将它们纯化到均匀（使用标准色谱方法），其数量与结晶方案兼容。有了这些知识，我们将尝试纯化一种非常相关的细胞质肌肉蛋白，骨骼肌二氢吡啶受体（DHPR）的β -1a亚基，由于其合成的限制，其结构尚未用原子分辨率确定。在特异性目标2中，我们将首先用编码荧光标记的跨膜蛋白的质粒电穿孔肌肉，并研究这些蛋白的哪种观察到的表达模式导致有效的膜蛋白提取方案。我们将重点关注以下整体膜蛋白（离子通道）的表达：骨骼肌DHPR的α -15亚基，Shaker K通道和哺乳动物骨骼ryanodine受体通道。随后，质粒将被设计用于表达6his标记的膜蛋白，以确保每个质粒都可以被分离和纯化到足够数量的均匀性，以允许它们结晶。

DESCRIPTION (provided by applicant): This R21 exploratory research grant responds to the NIGMS call for the development of novel eukaryotic expression systems capable of generating pure membrane proteins in sufficient quantities to afford crystallization trials. The lack of such a system has critically limited the number of structural studies of integral membrane proteins, such as ion channels and transporters, with atomic level resolution. The current application aims to cope with this problem by investigating the potential use of mammalian skeletal muscle to produce large quantities of recombinant membrane proteins when transfected in vivo with genetically engineered DNA plasmids. We propose that skeletal muscle may be an ideal preparation not only for the massive synthesis of cytosolic proteins, but most importantly, for the production and exportation of large quantities of transmembrane proteins. We plan to pursue these ideas experimentally in two separate, but intrinsically connected specific aims. Specific Aim 1 will test whether in vivo electroporation of muscle tissue with piasmids engineered for mammalian expression of six-histidine (6His) tagged EGFP and ECFP allows us to purify them to homogeneity (using standard chromatographic methods) in quantities compatible with crystallization protocols. With this knowledge, we will attempt to purify a very relevant cytosolic muscle protein, the beta-1a subunit of the skeletal muscle dihydropyridine receptor (DHPR), whose structure has not yet being determined with atomic resolution because of limitations in its synthesis. In Specific Aim 2, we will first electroporate muscle with plasmids encoding fluorescently-tagged transmembrane proteins and invest gate which of the observed expression patterns for these proteins leads to an efficient membrane protein extraction protocol. We will focus on the expression of the following integral membrane proteins (ionic channels): the alpha-15 subunit of the skeletal muscle DHPR, the Shaker K channel, and the mammalian skeletal ryanodine receptor channel. Later on, plasmids will be engineered for the expression of 6His-tagged membrane proteins in order to ensure that each of them can be isolated and purified to homogeneity in sufficient amount to permit their crystallization.