Protein Expression and Purification

摘要 蛋白质表达和纯化核心将为所有计划项目调查人员提供服务。核心B将表示 和纯化项目1、2、3和4的蛋白质。核心主任Sylvie Doublié博士拥有25年以上的经验 在蛋白质的表达和纯化方面。她将监督核心的蛋白质生产方面，并直接 监督负责蛋白质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，表达良好并具有活性。里克·伍德博士， 联席主任，在真核表达系统中表达人类蛋白质方面拥有丰富的经验。 他的团队是第一个表达全长、活性人类POLθ的小组。 核心B将为项目1、2、3和4生产可溶蛋白质及其变体。蛋白质结构将是 在不同的大肠杆菌菌株中表达，使用程序和方法，如自动诱导，已经证明 成功。核心还将优化蛋白质和复合体的溶解度和稳定性，用于 生化和细胞分析，结晶实验和单分子研究，通过表征 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。饱满 LENGTH POLθ和其他不能在大肠杆菌中表达的人修复因子将在人293中表达 细胞。 Core具备良好的表达和纯化蛋白质的能力。核心B也装备用于组织培养工作， 有必要的保温箱、引擎盖、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，因为 一个完整的。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实现目标，并进行统一的质量控制。核心B不复制任何大学核心 我们的研究团队可以使用的设施。

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.