Protein and Biochemistry

项目摘要（参见说明）： 蛋白质和生物化学核心将为该计划的所有成员表达和纯化蛋白质。 此外。 Core B 将进行蛋白质-DNA 相互作用的定量分析并表征运动学特征 使用高通量荧光测定法检测 DNA 修复蛋白的特性。 具体目标 1：(A) 优化 DNA 修复蛋白的表达和纯化 核心 B 将为项目 1、2、3 和 4 生产毫克量的可溶性蛋白质。我们将表达蛋白质 使用过去四年中已被证明成功的方案和方法在不同的大肠杆菌菌株中进行 我们实验室的，比如自诱导。我们将继续使用有限的蛋白水解 生物信息学来描绘功能域并表达更小的片段，如果全长蛋白质 构建体无法过度表达。 (B) 优化蛋白质和复合物的溶解度和稳定性 用于结晶实验，通过使用以下方法表征溶剂对蛋白质聚集特性的影响 动态光散射和分析凝胶过滤。将建立使用商业试剂盒的结晶试验 使用机器人工作站。 具体目标 2：对蛋白质-DNA 相互作用和稳态酶进行快速定量分析 本计划项目中产生的野生型和突变型 DNA 修复酶的动力学特性，使用 高通量荧光分析

PROJECT SUMMARY (See Instructions): The Protein and Biochemistry Core will express and purify proteins for all members of the Program Project. In addition. Core B will perform quantitative analyses of protein-DNA interacfions and characterize the kinefic properties of DNA repair proteins using high-throughput fluorimetric assays. Specific Aim 1: (A) To opfimize the expression and purificafion of DNA repair proteins Core B will produce mg amounts of soluble proteins for Projects 1, 2, 3, and 4. We will express the proteins in different E. coli strains using protocols and methods that have proven successful in the past four years in our laboratory, such as autoinducfion. We will continue to use limited proteolysis in conjunction with bioinformatics to delineate funcfional domains and express smaller fragments, if the full-length protein construct fails to overexpress. (B) To optimize the solubility and stability of proteins and complexes to be used in crystallizafion experiments, by characterizing solvent effects on protein aggregafion properties using dynamic light scattering and analytical gel filtrafion. Crystallization trials using commercial kits will be set up using a robofic workstafion. Specific Aim 2: To perform rapid quantitative analyses of protein-DNA interacfions and steady-state enzyme kinetic properties of wild-type and mutant DNA repair enzymes generated in this Program Project, using high-throughput fluorimetric assays