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。 亚拉巴马对伯明翰。蛋白质表达组将使用获得的专业知识作为核心膜 最近Mtb膜蛋白结构基因组学计划（GM 64676）的蛋白表达设施 为这一新的3个项目生成膜蛋白充分表达的方案， 程序.前一个方案有两个表达设施;一个在前苏联，一个在弗吉尼亚大学。综合这些 从350个Mtb开放阅读框中表达了203个内膜蛋白。外膜 这些蛋白质将在伯明翰的亚拉巴马大学进行表达和功能分析， Niederweis的专业知识。在重组表达外膜蛋白方面的额外专业知识， 来自Marassi和Opella的Marassi和内膜蛋白将补充表达技能， 核心A设施。该机构还将为该计划采购抗体。 目的1：全长膜蛋白的克隆、表达和纯化-所有膜蛋白 初始靶列表上的蛋白质已经被克隆并且大多数被过表达。新的克隆体将作为 这是将表达靶向特定细胞级分所必需的。已经制定了协议， 根据需要进行精制，以表达足够数量的蛋白质，用于项目1、2和3的研究。 纯化方案也在进行中，并将根据需要针对特定目标进行优化。 目标2.膜蛋白结构域和突变体的克隆、表达和纯化 全长蛋白的表达是第一要务，越来越多的证据表明，跨膜结构域 和膜外结构域通常独立发挥功能，因此一些蛋白质结构和 测定工作可以利用蛋白质结构域构建体。此外，将由该核心进行诱变 以稳定结构域或将Cys残基插入到原本无Cys的构建体中， 顺磁弛豫效应的限制，这将是重要的结构努力。 目标3.统一的同位素标记和氨基酸特异性标记-对于项目2和3是必不可少的。 将需要统一的15 N、13 C和2 H标记以及氨基酸特异性标记（标记一个 一种氨基酸，如缬氨酸）尽可能多的氨基酸。有效和经济的方法， 同位素标记将被使用和完善。 目标4。蛋白质的初步表征-凝胶电泳、圆二色性、光散射， 分析超离心和尺寸排阻色谱法将用于表征 样品制备物的均匀性和稳定性、二级结构含量和低聚物 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.