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

• 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
• 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

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