Core B. Biochemistry and Enzymology

核心 B. 生物化学和酶学

• 批准号: 10641863
• 负责人: JAMES C SACCHETTINI
• 金额: $ 13.56万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-12 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10641863
• 关键词: Adopted; Binding; Biochemical; Biochemistry; Bioinformatics; Biological; Biological Assay; Biology; Cloning; Collaborations; Disease; Enzymatic Biochemistry; Enzymes; Equipment; Genetic; Goals; In Vitro; Individual; Laboratories; Lead; Methods; Molecular; Mycobacterium tuberculosis; Open Reading Frames; Pathway Analysis; Pathway interactions; Proteins; Publishing; Reaction; Recombinant Proteins; Recombinants; Recording of previous events; Resources; Stress; Structure; Testing; Tuberculosis; Work; assay development; cofactor; design; enzyme activity; experience; insight; macromolecule; member; metabolomics; overexpression; pathogen; programs; protein expression; protein function; protein purification; structural genomics

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.

核心B，摘要 这个项目的总体目标是了解结核病生物学中重要的关键途径 在病原体的适应疾病相关的压力条件-包括从遗传和分子 表征到对功能的生物化学理解。最终，这需要纯化的蛋白质， 酶或结合测定。Sacchettini实验室在蛋白质表达方面有相当丰富的经验， 纯化以及开发生物化学测定以研究蛋白质功能。虽然他们通常 该方法以必需酶为研究对象，适用于大多数生物大分子。的 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，他们将提供结构资源，以帮助确定功能。他们 将生产重组蛋白，并为项目内的所有项目开发检测方法， 在实验室或出版的方法中开发。对于那些没有第一手资料或公开资料的情况， 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，他们已经从他们的研究中采用了这种策略。 用于高通量克隆、表达和纯化的结构基因组学管道。 核心在定义靶蛋白的生化功能方面的工作将在很大程度上依赖于与蛋白质的相互作用。 代谢组学核心（A核心）。这不会成为一个障碍，因为这些实验室目前正在密切合作， 项目此外，核心将使用标准的生物信息学方法来帮助了解氨基酸是什么。 除了类似蛋白质的结构之外，序列还提示了功能。核心将执行两个主要任务 活动： 活动1：克隆、过表达和纯化重组Mtb蛋白，用于在 体外生物化学表征和测定。 活动2：开发项目蛋白质的体外生物化学测定。