Protein and Biochemistry

项目总结（见说明）：蛋白质和生物化学核心将表达和纯化程序项目的所有成员的蛋白质。另外。核心B将进行蛋白质-DNA相互作用的定量分析，并使用高通量荧光测定法表征DNA修复蛋白的动力学特性。 具体目标1：（A）为了优化DNA修复蛋白的表达和纯化，核心B将产生mg量的可溶性蛋白，用于项目1、2、3和4。我们将在不同的E.大肠杆菌菌株使用的协议和方法，已被证明是成功的，在过去的四年中，在我们的实验室，如自诱导。我们将继续使用有限的蛋白水解结合生物信息学来描绘功能结构域和表达较小的片段，如果全长蛋白质构建未能过表达。(B)通过使用动态光散射和分析凝胶过滤表征溶剂对蛋白质聚集特性的影响，优化用于结晶实验的蛋白质和复合物的溶解度和稳定性。将使用机器人工作站设置使用商业试剂盒的结晶试验。 具体目标二：使用高通量荧光分析法对本项目中产生的野生型和突变型DNA修复酶的蛋白质-DNA相互作用和稳态酶动力学特性进行快速定量分析

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 interactions and characterize the kinetic properties of DNA repair proteins using high-throughput fluorimetric assays. Specific Aim 1: (A) To optimize the expression and purification 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 autoinduction. We will continue to use limited proteolysis in conjunction with bioinformatics to delineate functional 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 crystallization experiments, by characterizing solvent effects on protein aggregation properties using dynamic light scattering and analytical gel filtration. Crystallization trials using commercial kits will be set up using a robotic workstation. Specific Aim 2: To perform rapid quantitative analyses of protein-DNA interactions and steady-state enzyme kinetic properties of wild-type and mutant DNA repair enzymes generated in this Program Project, using high-throughput fluorimetric assays