Core D1: Membrane Protein Expression/Purification

蛋白质核心的主要作用将是为桥梁和试点项目的每个项目生产蛋白质。 此外，正在寻求帮助开发特定膜蛋白的高效表达、洗涤剂增溶优化和蛋白质目标纯化的方法的财团外的研究人员将可以访问Core。核心的重点将是制备用于结构、动力学和机制研究的均一功能蛋白质。核心D1成员将合作，利用他们的集体专业知识，为联盟使用的蛋白质开发和优化新的表达方案。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，包括在细胞中或无细胞中的原核和真核表达，以适应每种蛋白质靶标的特殊要求。因为没有一项拟议的研究能够完成 在没有蛋白质的情况下，我们将从这个项目开始就使用我们的技术尽快为财团提供样品。 总体而言，膜蛋白在结构确定的每一步都面临挑战。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伴侣[1，2]。在细菌中的表达受到青睐，因为通过发酵大量培养的成本很低， 高产量的潜力、非常快的生长速度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往往阻碍了足够数量的蛋白质以适当的折叠方式表达，而且可能不可能在均一状态下分离和纯化足够数量的蛋白质。出于这些原因，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，这应该提供任何选定的蛋白质靶标可以产生的高概率。此外，我们还描述了进一步增强和优化表达的高通量筛选方法。因为该联盟强调对蛋白质的研究，对于这些蛋白质，高分辨率结构是 目前已经成功地实现了这类蛋白的高水平表达，并制备了均一的纯化蛋白。我们将利用已有的信息，但在某些情况下，建议研究目标，特别是尚未在高水平表达的突变形式。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，蛋白质可能是复合体的一部分，这需要表达多种蛋白质，其中一些蛋白质在细菌中不表达。这个例子可能需要在哺乳动物或昆虫细胞中同时表达所有成分以进行适当的处理。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、纯化程度和操作所需的修改。最重要的是蛋白质的功能。出于这个原因，蛋白质核心将与所有调查人员密切合作，以确保每个目标的活性得到适当的评估。

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.