Mammalian skeletal muscle: a recombinant protein factory

描述（由申请人提供）：该R21探索性研究补助金响应NIGMS呼吁开发能够产生足够数量的纯膜蛋白以进行结晶试验的新型真核表达系统。缺乏这样一个系统，严重限制了一些完整的膜蛋白，如离子通道和转运蛋白的结构研究，原子水平的分辨率。本申请旨在通过研究哺乳动物骨骼肌在用遗传工程DNA质粒体内转染时产生大量重组膜蛋白的潜在用途来科普这个问题。我们建议，骨骼肌可能是一个理想的准备，不仅为大量合成的胞质蛋白，但最重要的是，为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具体目标中实验性地追求这些想法。具体目标1将测试是否在体内电穿孔的肌肉组织与质粒工程哺乳动物表达的六组氨酸（6 His）标记的EGFP和ECFP允许我们纯化它们的同质性（使用标准色谱方法），在数量上与结晶协议兼容。有了这些知识，我们将试图纯化一个非常相关的胞质肌肉蛋白，β-1a亚基的骨骼肌二氢吡啶受体（DHPR），其结构尚未确定与原子分辨率，因为其合成的限制。在特定目标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，并研究观察到的这些蛋白的表达模式中的哪一种导致有效的膜蛋白提取方案。我们将重点关注以下膜蛋白（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肌ryanodine受体通道。随后，质粒将被工程化用于表达6 His标记的膜蛋白，以确保它们中的每一个可以被分离并纯化至足够量的同质性以允许它们的结晶。

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.