Protein Expression and Antibody Development

蛋白质表达核心设施将设在佛罗里达州立大学和佛罗里达大学 阿拉巴马州的伯明翰。蛋白质表达小组将利用所获得的专业知识作为核心膜 最新Mtb膜蛋白结构基因组学计划(GM64676)的蛋白质表达设施 为这一新的3个项目生成充分表达膜蛋白的方案 程序。前一个项目有两个表达设施；一个在佛罗里达州立大学，另一个在弗吉尼亚大学。把这些放在一起 设备从350个Mtb的开放阅读框中表达了203个内膜蛋白。外膜 蛋白质将在阿拉巴马大学伯明翰分校进行表达和功能分析，以利用 尼德韦斯的专业知识。在重组表达外膜蛋白方面的其他专业知识 Marassi和在内膜蛋白Marassi和Opella将补充表达技能 核心A设施。该机构还将为该项目采购抗体。 目的1：全长膜蛋白的克隆、表达和纯化 最初的目标列表上的蛋白质已经被克隆，并得到了大多数的过度表达。新的克隆将作为 必须针对特定的“细胞部分”来表达。已经制定了协议，并将 根据需要进行提纯，以表达足够数量的蛋白质，以供项目1、2和3的研究使用。 纯化方案也在手中，并将根据需要针对特定目标进行优化。 目的2.膜蛋白结构域和突变体的克隆、表达和纯化 表达全长蛋白是当务之急，越来越多的证据表明跨膜结构域 膜外结构域通常独立发挥作用，因此一些蛋白质结构和 化验工作可以利用蛋白质结构域。此外，诱变将由该核心执行 稳定结构域或在无半胱氨酸的结构中插入半胱氨酸残基，以使能够收集 顺磁松弛效应约束，这将是重要的结构努力。 目的3.统一的同位素标记和氨基酸特异性标记是项目2和项目3所必需的。 将需要统一的15N、13C和2H标记以及特定氨基酸的标记(一个标记 氨基酸的类型，如缬氨酸)，尽可能多的氨基酸。有效且经济的方法 将使用和改进同位素标记。 目的4.蛋白质的初步表征--凝胶电泳、圆二色谱、光散射、 分析超速离心法和尺寸排除层析法将用于表征 样品制备、二级结构含量和齐聚物的均一性和稳定性 蛋白质的状态。

The Protein Expression Core Facility will be located at Florida State University and the University of Alabama at Birmingham. The protein expression group will use the expertise gained as a Core Membrane Protein Expression Facility for the recent Mtb Membrane Protein Structural Genomics Program (GM64676) to generate protocols for the sufficient expression of membrane proteins for all 3 Projects of this new Program. The former Program had two expression facilities; one at FSU and one at the UVa. Together these facilities expressed 203 inner membrane proteins from 350 Mtb open reading frames. Outer membrane proteins will be expressed and functionally analyzed at the University of Alabama at Birmingham to utilize the expertise of Niederweis. Additional expertise in the recombinant expression of outer membrane proteins from Marassi and in inner membrane proteins from Marassi and Opella will complement the expression skills of the Core A Facility. This Facility will also procure antibodies for the Program. Aim 1: Cloning, expression, and purification of full length membrane proteins -All of the membrane proteins on the Initial Target List have been cloned and most over-expressed. New clones will be made as necessary to target the expression to particular'cell fractions. Protocols have been developed and will be refined as necessary to express the proteins in sufficient quantities for the research in Projects 1, 2 and 3. Purification protocols are also in hand and will be optimized for the specific targets, as necessary. Aim 2. Cloning, expression, and purification of membrane protein domains and mutants - While expression of full-length proteins is the first priority, there is growing evidence that transmembrane domains and extra-membranous domains often function independently and therefore some protein structural and assay efforts could utilize protein domain constructs. In addition, mutagenesis will be performed by this Core to stabilize domains or to insert Cys residues in an otherwise Cys-less construct to enable the collection of paramagnetic relaxation effect restraints that will be important for the structural efforts. Aim 3. Uniform isotopic labeling and amino acid specific labeling - are essential for Projects 2 and 3. Uniform 15N, 13C and 2H labeling will be required as well as amino acid specific labeling (labeling of one type of amino acid, such as valine) of as many amino acids as possible. Effective and economic methods for isotope labeling will be used and refined. Aim 4. Initial characterization of the proteins - Gel electrophoresis, circular dichroism, light scattering, analytical ultracentrifugation, and size exclusion chromatography will be used to characterize the homogeneity and stability of the sample preparations, the secondary structure content, and the oligomeric state of the protein.