Protein Expression and Purification

蛋白质表达和纯化

• 批准号: 10468634
• 负责人: Sylvie Doublie
• 金额: $ 19.06万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10468634
• 关键词: Adherent Culture; Affinity Chromatography; Back; Biochemical; Biochemistry; Biophysics; Cell Line; Cells; Cellular Assay; Charge; Core Facility; Crystallization; DNA-Directed DNA Polymerase; Engineering; Escherichia coli; Gel Chromatography; Genomic Instability; Goals; Human; Human Resources; Incubators; Individual; Ion Exchange; Length; Liquid substance; Malignant Neoplasms; Mammalian Cell; Mediating; Methods; Microscope; Molecular; Molecular Biology; Play; Polymerase; Production; Property; Proteins; Proteolysis; Protocols documentation; Quality Control; Reagent; Research; Research Personnel; Robotics; Role; Secure; Sequence Alignment; Services; Site; Sodium Chloride; Solubility; Solvents; Supervision; Suspension Culture; System; Tertiary Protein Structure; Universities; Variant; Wood material; Work; cost effective; experience; experimental study; human DNA; in silico; light scattering; programs; protein aggregation; protein complex; protein expression; protein purification; repaired; single molecule; structural biology; success; tissue culture; vector

SUMMARY The Protein Expression and Purification Core will serve all Program Project investigators. Core B will express and purify proteins for Projects 1, 2, 3, and 4. The Core Director, Dr. Sylvie Doublié, has 25+ years experience in protein expression and purification. She will oversee the protein production aspect of the Core and directly supervise the staff in charge of protein expression and purification. Her lab was the first to engineer a construct of the polymerase domain of human DNA Polymerase θ that expresses well and is active. Dr. Rick Wood, the Co-Director, has extensive experience in the expression of human proteins in eukaryotic expression systems. His group was the first to express full-length, active human Pol θ. Core B will produce soluble proteins and their variants for Projects 1, 2, 3, and 4. Protein constructs will be expressed in different E. coli strains using protocols and methods, such as autoinduction, that have proven success. The Core will also optimize the solubility and stability of proteins and complexes to be used in biochemical and cellular assays, crystallization experiments and single-molecule studies, by characterizing solvent effects on protein aggregation properties using dynamic light scattering and analytical gel filtration. Full length Pol θ and other human repair factor that cannot be expressed in E. coli will be expressed in human 293 cells. The Core is well equipped to express and purify proteins. Core B is also equipped for tissue culture work, having the necessary incubators, hoods, microscope, centrifuge, and storage facilities. The services provided by Core B are essential for each Project and for the success of the Program Project as a whole. The Protein Expression and Purification Core will allow the different projects to achieve their research goals in a cost-effective manner and with unified quality control. Core B does not duplicate any University core facilities that would be available to our research team.

总结 蛋白质表达和纯化核心将为所有项目研究人员提供服务。核心B将表达 并纯化项目1、2、3和4的蛋白质。核心总监Sylvie Doublié博士拥有25年以上的经验 蛋白质表达和纯化。她将监督核心的蛋白质生产方面， 监督负责蛋白表达和纯化的工作人员。她的实验室是第一个设计出 人DNA聚合酶θ的聚合酶结构域，表达良好且有活性。里克·伍德博士 Co-Director，在真核表达系统中表达人类蛋白质方面拥有丰富的经验。 他的小组是第一个表达全长、活性人类Pol θ的小组。 核心B将为项目1、2、3和4产生可溶性蛋白及其变体。蛋白质构建体将是 在不同的E.大肠杆菌菌株使用的协议和方法，如自诱导，已证明 成功核心还将优化蛋白质和复合物的溶解度和稳定性，以用于 生物化学和细胞分析，结晶实验和单分子研究，通过表征 使用动态光散射和分析凝胶过滤研究溶剂对蛋白质聚集特性的影响。充分 长度Pol θ和其它不能在E.将在人293中表达 细胞 核心是装备精良，表达和纯化蛋白质。核心B还配备了组织培养工作， 具有必要的培养箱、通风橱、显微镜、离心机和储存设施。 核心B提供的服务对每个项目和计划项目的成功至关重要， 整体蛋白质表达和纯化核心将允许不同的项目实现他们的研究 以具有成本效益的方式和统一的质量控制来实现目标。核心B不重复任何大学核心 我们的研究团队可以使用的设施。