Protein and Biochemistry

项目摘要(见说明)：蛋白质和生物化学核心将为计划项目的所有成员表达和纯化蛋白质。此外。Core B将对蛋白质-DNA相互作用进行定量分析，并使用高通量荧光分析方法表征DNA修复蛋白质的动力学特性。 具体目标1：(A)为了优化DNA修复蛋白的表达和纯化，Core B将为项目1、2、3和4生产mg量的可溶性蛋白。我们将使用过去四年在我们实验室被证明成功的方案和方法在不同的大肠杆菌菌株中表达这些蛋白，例如自动诱导。如果全长蛋白质结构不能过度表达，我们将继续使用有限的蛋白质分解与生物信息学相结合来描绘功能结构域并表达较小的片段。(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体的溶解度和稳定性。使用商业工具包的结晶试验将使用机器人工作站进行。 具体目标2：使用高通量荧光分析技术，对本项目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