Core B. Biochemistry and Enzymology

核心 B. 生物化学和酶学

• 批准号: 10190811
• 负责人: JAMES C SACCHETTINI
• 金额: $ 12.93万
• 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/10190811
• 关键词: Adopted; Binding; Biochemical; Biochemistry; Bioinformatics; Biological; Biological Assay; Biology; Cloning; Collaborations; Crystallization; 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 该计划项目的总体目标是了解 Mtb 生物学中重要的关键途径 病原体对疾病相关应激条件的适应 - 包括从遗传和分子 表征到对功能的生化理解。最终这需要纯化的蛋白质和 酶促或结合测定。 Sacchettini 实验室在蛋白质表达和 纯化以及开发生化测定来研究蛋白质功能。虽然他们通常 重点关注必需酶，所用方法可适用于大多数生物大分子。这 Sacchettini 实验室还充当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 将利用方法生产重组蛋白并为该计划项目内的所有项目开发检测方法 在实验室中开发或已发布的方法。对于没有第一手或公开信息的情况， 他们将使用他们的一般策略来制造可溶性和活性的结核分枝杆菌蛋白，这是他们从他们的研究中采用的 用于高通量克隆、表达和纯化的结构基因组学管道。 核心在定义目标蛋白的生化功能方面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在几个方面密切合作 项目。此外，核心将使用标准生物信息学方法来帮助了解氨基的含义。 除了类似蛋白质的结构之外，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、过表达和纯化重组 Mtb 蛋白，用于功能分析 体外生化表征和测定。 活动 2：开发项目蛋白的体外生化测定。