Core D1: Membrane Protein Expression/Purification

蛋白质核心的主要作用是为每个桥梁和试点项目生产蛋白质。 此外，该核心将可供财团以外的研究人员使用，他们正在寻求帮助，以开发特定膜蛋白的高产表达，去污剂溶解优化和蛋白质靶点纯化的方法。核心的重点将是制备同质功能蛋白质用于结构，动力学和机制的研究。核心D1成员将利用他们的集体专业知识合作开发和优化联盟使用的蛋白质的新表达方案。核心将利用其现有的专业知识和技术，提供广泛的表达系统，包括原核和真核细胞表达或无细胞表达，以适应每种蛋白质靶标的特定要求。因为没有一项研究能够完成 如果没有蛋白质，我们将使用我们的技术，从这个项目开始，尽快为财团提供样品。 一般来说，膜蛋白在结构确定的每一步都存在挑战。这些蛋白质的合成和加工是复杂的，通常涉及特定的折叠因子或分子伴侣[1，2]。在细菌中的表达是有利的，因为通过发酵生长大培养体积的成本低， 高产潜力、非常快的生长速率以及表达系统的简单性和灵活性。 然而，蛋白质加工方式的内在差异通常会阻止表达足够量的具有适当折叠的蛋白质，并且可能无法以均质状态分离和纯化足够量的蛋白质。由于这些原因，蛋白质核心将采用一系列表达系统，包括 制备规模的无细胞系统，其应该提供可以产生任何选择的蛋白质靶标的高概率。此外，我们描述了高通量筛选方法，以进一步增强和优化表达。因为该联盟强调研究的蛋白质的高分辨率结构， 这些蛋白质已经成功地高水平表达，并制备了均一的纯化蛋白质。我们将利用现有的信息，但在某些情况下，提出了研究的目标，特别是突变形式，尚未在高水平上表达。此外，研究人员可能需要特定环境或状态下的蛋白质靶标。为 例如，蛋白质可以是复合物的一部分，其需要多种蛋白质的表达，其中一些蛋白质在细菌中不表达。该实施例可能需要在哺乳动物或昆虫细胞中同时表达所有组分以进行适当处理。蛋白质核心将应用一系列表达策略 考虑到几个需要：数量、多聚体状态、纯化水平和操作所需的修饰。最重要的是蛋白质的功能。因此，Protein Core将与所有研究人员密切合作，以确保正确评估每个靶标的活性。

The main role of the Protein Core will be to produce proteins for each of the Bridge and Pilot Projects. Furthermore, the Core will be accessible to investigators outside the consortium who are seeking help to develop methods for high yield expression of specific membrane proteins, detergent solubilization optimization, and purification of protein targets. The emphasis of the Core will be preparation of homogeneous functional proteins for use in studies of structure, dynamics and mechanism. Core D1 members will cooperate in using their collective expertise to develop and optimize new expression protocols for proteins used by the Consortium. The Core will utilize its expertise and technologies already in place to provide a wide range of expression systems, both prokaryotic and eukaryotic expression in cells or cell-free, to accommodate the specific requirements of each protein target. Because none of the proposed studies can be accomplished without protein, we will use our technologies from the onset of this project to provide samples for the Consortium as soon as possible. In general, membrane proteins present challenges at every step of the way to structural determination. The synthesis and processing of these proteins is complex and often involves specific folding factors or chaperones [1, 2]. Expression in bacteria is favored because of the low cost to grow large culture volumes by fermentation, the potential for high yields, the very fast growth rate, and the simplicity and flexibility of expression systems. However, intrinsic differences in how proteins are processed often prevent the expression of adequate amounts of protein with the proper fold, and it may not be possible to isolate and purify sufficient quantities in a homogenous state. For these reasons, the Protein Core will employ a range of expression systems including preparative scale cell-free systems, which should provide a high probability that any selected protein target can be produced. In addition, we describe high throughput screening methods to further enhance and optimize expression. Because the Consortium emphasizes the study of proteins for which a high resolution structure is already accomplished, such proteins have already been successfully expressed at high levels and homogeneous purified protein prepared. We will take advantage of the information already available, but in some cases targets are proposed for study, in particular mutant forms, that have not been expressed at high levels. Furthermore, investigators may require the protein target in a specific environment or state. For example, the protein may be part of a complex, which requires expression of multiple proteins, some of which do not express in bacteria. This example may require simultaneous expression of all the components in a mammalian or insect cell for proper processing. The Protein Core will apply a range of expression strategies taking into consideration several needs: the quantity, the multimeric state, the level of purification, and the modifications required for manipulations. Of foremost importance is the functionality of the protein. For this reason, the Protein Core will work closely with all investigators to assure that the activity of each target is properly assessed.