Protein Expression and Purification

蛋白质表达和纯化

基本信息

批准号：
10640913
负责人：
Sylvie Doublie
金额：
$ 18.74万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10640913
关键词：
Adherent Culture Affinity Chromatography Biochemical Biochemistry Biophysics Cell Line Cells Cellular Assay Charge Complex Core Facility Crystallization DNA-Directed DNA Polymerase Dedications Engineering Escherichia coli Gel Chromatography Genomic Instability Goals Human Human Resources Incubators Individual Ion Exchange Length Liquid substance Malignant Neoplasms Mammalian Cell Mediating Methods Microscope Molecular Molecular Biology Play Polymerase Production Property Proteins Proteolysis Protocols documentation Quality Control Reagent Research Research Personnel Robotics Role Secure Sequence Alignment Services Site Sodium Chloride Solubility Solvents Suspension Culture System Tertiary Protein Structure Universities Variant Wood material Work cost effective experience experimental study human DNA in silico light scattering programs protein aggregation protein expression protein purification repaired single molecule structural biology success tissue culture vector

项目摘要

SUMMARY The Protein Expression and Purification Core will serve all Program Project investigators. Core B will express and purify proteins for Projects 1, 2, 3, and 4. The Core Director, Dr. Sylvie Doublié, has 25+ years experience in protein expression and purification. She will oversee the protein production aspect of the Core and directly supervise the staff in charge of protein expression and purification. Her lab was the first to engineer a construct of the polymerase domain of human DNA Polymerase θ that expresses well and is active. Dr. Rick Wood, the Co-Director, has extensive experience in the expression of human proteins in eukaryotic expression systems. His group was the first to express full-length, active human Pol θ. Core B will produce soluble proteins and their variants for Projects 1, 2, 3, and 4. Protein constructs will be expressed in different E. coli strains using protocols and methods, such as autoinduction, that have proven success. The Core will also optimize the solubility and stability of proteins and complexes to be used in biochemical and cellular assays, crystallization experiments and single-molecule studies, by characterizing solvent effects on protein aggregation properties using dynamic light scattering and analytical gel filtration. Full length Pol θ and other human repair factor that cannot be expressed in E. coli will be expressed in human 293 cells. The Core is well equipped to express and purify proteins. Core B is also equipped for tissue culture work, having the necessary incubators, hoods, microscope, centrifuge, and storage facilities. The services provided by Core B are essential for each Project and for the success of the Program Project as a whole. The Protein Expression and Purification Core will allow the different projects to achieve their research goals in a cost-effective manner and with unified quality control. Core B does not duplicate any University core facilities that would be available to our research team.

概括蛋白质表达和纯化核心将为所有计划项目核心 B 的研究人员提供服务。并为项目 1、2、3 和 4 纯化蛋白质。核心总监 Sylvie Doublié 博士拥有 25 年以上的经验她将直接监督核心的蛋白质生产方面。监督负责蛋白质表达和纯化的工作人员，她的实验室是第一个设计构建体的实验室。人类 DNA 聚合酶 θ 的聚合酶结构域表达良好且具有活性。联合主任，在真核表达系统中表达人类蛋白质方面拥有丰富的经验。他的团队是第一个表达完整长度、活跃的人类 Pol θ 的团队。核心 B 将为项目 1、2、3 和 4 生产可溶性蛋白质及其变体。蛋白质构建体将是使用已证明的自诱导等方案和方法在不同的大肠杆菌菌株中表达该核心还将优化所用蛋白质和复合物的溶解度和稳定性。生化和细胞测定、结晶实验和单分子研究，通过表征使用动态光散射和分析凝胶过滤，溶剂对蛋白质聚集特性的影响。长度 Pol θ 和其他无法在大肠杆菌中表达的人类修复因子将在人类 293 中表达细胞。 Core 非常适合表达和纯化蛋白质，Core B 也适合组织培养工作。拥有必要的培养箱、通风橱、显微镜、离心机和储存设施。核心 B 提供的服务对于每个项目以及计划项目的成功至关重要，因为蛋白质表达和纯化核心将使不同的项目能够实现他们的研究。核心 B 不重复任何大学核心我们的研究团队可以使用的设施。