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汇集的 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 该计划在过去10年中已成功实施，并且成效卓著。这 集中资源提供分子生物学、细菌/酵母表达和 制备过表达维生素K依赖性蛋白、因子V变体 和因子VIII变体。核心为大规模蛋白质生产提供设施、硬件和辅助 在无血清条件培养基中用稳定或瞬时转染的哺乳动物细胞进行生产。的 核心将提供大规模分离重组蛋白的设施，适当的色谱 策略，并将维护此活动所需的亲和列。核心还将提供一些 质量控制服务，特别是定量β-羧基谷氨酸含量，以验证完整性 表达维生素K依赖蛋白。还将为大规模的 从血浆中分离凝固蛋白。核心B人员将协助项目成员实施 稀疏矩阵晶体屏幕，联络与宾夕法尼亚大学x射线设施，使衍射数据的收集 提供专业知识，用于使用扩展筛优化结晶条件。 核心支持高性能计算机配备必要的程序处理衍射 数据，通过分子置换和细化建立模型。核心还将为小型 关于X射线角散射测量的源程序和数据分析。氢/氘交换 研究将与李仁浩合作进行，并通过与埃默里大学的适度分包合同来促进 大学主任将负责监督和有效开展这些活动，并 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