Protein and Biochemistry

蛋白质和生物化学

• 批准号: 8381911
• 负责人: Sylvie Doublie
• 金额: $ 22.14万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-03 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8381911
• 关键词: Bioinformatics; Biological Assay; Crystallization; DNA; DNA Repair Enzymes; DNA glycosylase; DNA repair protein; DNA-Protein Interaction; Enzyme Kinetics; Enzymes; Escherichia coli; Gel; Gel Chromatography; Genetic Recombination; Instruction; Laboratories; Length; Malignant Neoplasms; Methods; Play; Predisposition; Property; Protein Biochemistry; Proteins; Proteolysis; Protocols documentation; Role; Solubility; Solvents; Structure; Variant; cancer therapy; light scattering; member; mutant; overexpression; programs; protein aggregation; protein complex; research study

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

项目摘要（请参阅说明）： 蛋白质和生物化学核心将为该计划项目的所有成员表达和净化蛋白质。 此外。核心B将对蛋白质-DNA间隔进行定量分析，并表征动力学。 使用高通量荧光测定的DNA修复蛋白的性质。 特定目的1：（a）操作DNA修复蛋白的表达和净化蛋白的表达和净化 核心B将生成项目1、2、3和4的毫克可溶性蛋白。我们将表达蛋白质 在过去四年中，使用方案和方法在不同的大肠杆菌菌株中 我们的实验室，例如自动引导。我们将继续使用有限的蛋白水解结合 如果全长蛋白 构造无法过表达。 （b）优化蛋白质和复合物的溶解度和稳定性 通过使用使用溶剂对蛋白质聚集特性的溶剂作用来表征Crystallizafion实验 动态光散射和分析凝胶过滤。将使用商业套件进行结晶试验 使用Robofic Workstafion。 特定目的2：对蛋白质-DNA间隔和稳态酶进行快速定量分析 该程序项目中生成的野生型和突变DNA修复酶的动力学特性，使用 高通量荧光测定