Core B. Biochemistry and Enzymology

核心B，摘要 该项目的总体目标是了解结核分枝杆菌生物学中重要的关键途径。 在病原体对与疾病相关的应激条件的适应中-包括从遗传和分子上 从生物化学的角度来理解功能。最终，这既需要纯化的蛋白质，也需要 酶分析或结合分析。Sacchettini实验室在蛋白质表达方面拥有丰富的经验，并且 纯化以及正在开发的生化分析以研究蛋白质的功能。虽然他们通常 聚焦于关键酶，所使用的方法可以适用于大多数生物大分子。这个 Sacchettini实验室也是结核病结构基因组学联盟(TBSGC)的重要接口 在适当的时候，它们将提供对结构资源的访问，以帮助确定功能。他们 将生产重组蛋白，并利用方法，为计划项目内的所有项目开发分析 在实验室中开发或发布的方法。对于那些没有第一手或发布的信息的情况， 他们将使用他们的一般策略来制造可溶性和活性的Mtb蛋白，这是他们从他们的 高通量克隆、表达和纯化的结构基因组学流水线。 核心确定目标蛋白质的生化功能的工作将在很大程度上依赖于与 代谢组学核心(核心A)。这不会成为障碍，因为这些实验室目前正密切合作 项目。此外，Core将使用标准的生物信息学方法来帮助了解氨基酸 除了相似蛋白质的结构外，序列还暗示了功能。核心将执行两个主要任务 活动： 活动1：克隆、高效表达和纯化重组Mtb蛋白，用于功能分析 体外生化特性和检测。 活动2：建立工程蛋白的体外生化分析方法。

Core B, Abstract The overall goal of this program project is to understand key pathways in the biology of Mtb that are important in the pathogen’s adaptation to disease-relevant stress conditions – including moving from genetic and molecular characterization to a biochemical understanding of function. Ultimately this requires both purified proteins, and enzymatic or binding assays. The Sacchettini lab has considerable experience with both protein expression, and purification as well as with developing biochemical assays to investigate protein function. While they usually focus on essential enzymes, the methods used can be applied to most biological macromolecules. The Sacchettini laboratory also serves as an important interface to the TB Structural Genomics Consortium (TBSGC) and when appropriate they will provide access to structural resources in order to help determine function. They will produce recombinant protein and develop assays for all projects within the program project, utilizing methods developed in the lab or published methods. For those cases where there is no first hand or published information, they will use their general strategy for making soluble and active Mtb proteins, that they have adopted from their structural genomics pipeline for high throughout cloning, expression and purification. The Core’s work on defining the biochemical function of the target proteins will rely heavily on interactions with the metabolomics core (Core A). This will not be an obstacle as these labs currently work closely on several projects. In addition, the Core will use standard bioinformatic methods to help understand what the amino sequence suggests about function, in addition to structures of similar proteins. The core will perform two main activities: Activity 1: Clone, overexpress and purify recombinant Mtb proteins for functional analysis using in vitro biochemical characterization and assays. Activity 2: Develop in vitro biochemical assays for project proteins.