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蛋白质的程序项目的所有成员。 另外。核心B将对蛋白质-DNA相互作用进行定量分析，并表征 使用高通量荧光测定的DNA修复蛋白的性质。 具体目的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产生mg量的可溶性蛋白。我们将表达蛋白质 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，在过去四年中， 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 生物信息学描绘功能域和表达较小的片段，如果全长蛋白质 构建体不能过表达。(B)为了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 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 使用机器人工作人员。 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