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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过去十年来，这一倡议得到了成功实施，并且非常富有成效。这集中资源提供分子生物学、细菌/酵母表达和过表达维生素K依赖性蛋白、因子V变体的稳定哺乳动物细胞系的制备和因子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