Core B: Molecular Biology, Protein Expression and Structural Biology

核心B摘要 分子生物学、蛋白质表达和结构生物学核心是围绕着 项目负责人在项目中的资源和专业知识。这个核心的行为是基于一个模型 这在过去10年中得到了成功实施，并取得了极大的成效。这 集中资源提供分子生物学、细菌/酵母表达以及 稳定高表达维生素K依赖蛋白和因子V变异体的哺乳动物细胞系的建立 以及因子VIII的变异体。核心为大规模蛋白质提供设施、硬件和辅助 用稳定的或瞬时转基因的哺乳动物细胞在无血清条件培养液中进行生产。这个 核心将为大规模分离重组蛋白提供设施，进行适当的层析 战略，并将维护本活动所需的亲和力栏目。核心还将提供一些 质量控制服务，特别是-羧基谷氨酸含量的定量，以验证 表达维生素K依赖蛋白。还将为大型项目提供援助和设施 从血浆中分离凝血蛋白。核心B人员将协助计划成员实施 稀疏矩阵晶体屏幕，与宾夕法尼亚大学的x射线设施联系，以允许收集衍射数据 该公司提供国内来源，并提供专业技术，用于优化扩大筛网的结晶条件。 该核心支持配备了处理衍射所需程序的高性能计算机 数据，通过分子替换和精化来建立模型。核心还将为小型企业提供支持 Home源的角度x射线散射测量和数据分析。氢/氢交换 研究将与李仁浩合作进行，并通过与埃默里公司签订一份适度的分包合同来促进 大学。主任将负责监督和有效开展这些活动，并负责 确保为项目提供公平的服务

Core B Abstract The molecular biology, protein expression and structural biology core is organized around the pooling of the resources and expertise of the project leaders in the program. The conduct of this core is based on a model that has been successfully implemented in the past 10 years and has been extremely productive. This centralized resource provides service and assistance in molecular biology, bacterial/yeast expression and in the preparation of stable mammalian cell lines over-expressing vitamin K-dependent proteins, factor V variants and factor VIII variants. The core provides the facilities, hardware and assistance for large-scale protein production runs with stable or transiently transfected mammalian cells in serum-free conditioned media. The core will provide facilities for large scale isolation of recombinant proteins, the appropriate chromatography strategies and will maintain the affinity columns necessary for this activity. The core will also provide some quality control services particularly the quantitation of -carboxyglutamic acid content to verify the integrity of expressed vitamin K-dependent proteins. Assistance and facilities will also be provided for the large-scale isolation of coagulation proteins from plasma. Core B personnel will assist program members in implementing sparse matrix crystal screens, liaise with the UPenn x-ray facility to allow the collection of diffraction data on the home source and provide expertise for optimization of crystallization conditions with expanded screens. The core supports high performance computers equipped with necessary programs for processing diffraction data, model building by molecular replacement and refinement. The core will also provide support for small angle x-ray scattering measurements on the Home source and data analysis. Hydrogen/deuterium exchange studies will be performed in collaboration with Renhao Li and facilitated by a modest sub-contract to Emory University. The Director will be responsible for the oversight and efficient conduct of these activities and for ensuring that equitable service is provided to the projects