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Core B: Molecular Biology, Protein Expression and Structural Biology

核心 B：分子生物学、蛋白质表达和结构生物学

基本信息

批准号：
10175002
负责人：
Sriram Krishnaswamy
金额：
$ 65.52万
依托单位：
CHILDREN'S HOSP OF PHILADELPHIA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10175002
关键词：
Acids Affinity Area Awareness Biology Cell Line Chemistry Chromatography Coagulation Process Collaborations Collection Contracts Core Facility Crystallization Data Data Analyses Data Collection Deuterium Development Docking Ensure Factor V Factor VIII Feasibility Studies Goals Hemostatic function Home Human Resources Hydrogen Individual Laboratories Mammalian Cell Measurement Measures Modeling Molecular Molecular Biology Motivation Physiology Plasma Proteins Post-Translational Protein Processing Preparation Production Property Proteins Protocols documentation Quality Control Reaction Reagent Recombinant Proteins Recombinants Research Personnel Resolution Resources Roentgen Rays Running Schedule Series Serum Services Source Structure Synchrotrons Thrombosis Training Universities Variant Visit Vitamin K Work X ray diffraction analysis X-Ray Crystallography Yeasts base data modeling experience expression vector high end computer instrument interest liquid chromatography mass spectrometry member model building novel parallel computer programs protein expression protein purification repository skills stable cell line structural biology success

项目摘要

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

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