Protein Production Facility

蛋白质生产设施

基本信息

批准号：
7558822
负责人：
Robert Tse Nan Tjian
金额：
$ 23.48万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7558822
关键词：
Acute Adenoviruses Adopted Affinity Affinity Chromatography Amino Acids Animals Antibodies Antibody Affinity Antibody Formation Antigens Area Arts Avidity Back Baculoviruses Behavior Binding Biochemical Biochemistry Biological Assay Buffers CRSP7 gene CV-1 Cell Extracts Cell Line Cells Chromatin Chromatography Class Communication Complement Complementary DNA Complex Condition Consensus Core Facility Crude Extracts Crystallization Cultured Cells DNA Polymerase II DNA-Directed RNA Polymerase Detection Detergents Development Devices Disadvantaged Drosophila genus ERCC3 gene Embryo Endopeptidases Epitope Mapping Epitopes Equipment Eukaryotic Cell Facility Construction Funding Category Feedback Figs - dietary Fluorescence Resonance Energy Transfer Fractionation G-substrate Gel Chromatography Gene Expression Generations Genetic Transcription Glutathione S-Transferase Glycerol Group Meetings Hand Hela Cells High Pressure Liquid Chromatography Human Human Resources Hybridomas Hybrids In Vitro Individual Injection of therapeutic agent Institutes Ion Exchange Resins Joints Label Laboratories Learning Ligands Localized Location Maps Masks Mass Spectrum Analysis Membrane Methods Mind Molecular Molecular Cloning Molecular Conformation Molecular Machines Monitor Monoclonal Antibodies Mus N-terminal Nature Nuclear Nuclear Extract Numbers Peptide Hydrolases Peptide Mapping Peptide antibodies Peptides Phase Plant Resins Plasmids Positioning Attribute Preparation Principal Investigator Probability Procedures Production Property Protein Subunits Proteins Protocols documentation Purpose RNA RNA Polymerase II Reaction Reagent Recombinant Fusion Proteins Recombinant Proteins Recombinants Relative (related person)Research Personnel Roentgen Rays Route Sampling Scientist Screening procedure Series Set protein Sf9 cell line Simian virus 40 Site Sodium Chloride Solubility Solutions Solvents Sorting - Cell Movement Source Specialist Specificity Staging Staining method Stains Standards of Weights and Measures Structure Structure-Activity Relationship Study Subject Surface System TATA-Box Binding Protein Testing Time Transcription Factor TFIIA Translating Viral Western Blotting Wit Work X-Ray Crystallography analytical tool base cell type chromatin remodeling deletion analysis density design desire experience fly follow-up gene replacement in vivo member monoclonal antibody production novel particle phosphocellulose polypeptide programs promoter protein purification reconstitution reconstruction research and development research study single-molecule FRET stable cell line success three dimensional structure tool transcription factor transcription factor TFIIH user-friendly vector

项目摘要

A major impetus for developing this program project is to consolidate and coordinate our efforts to establish a core laboratory designed to facilitate the production and purification of large multi- subunit regulatory complexes involved in gene transcription. Consequently, the establishment of a highly efficient, well integrated and interdependent core facility to meet these considerable experimental challenges will be critical to the success of this Program Project. We have therefore subdivided the Core Lab into 3 distinct divisions, each with its own set of defined specific aims: 1) Construction of expression systems and production of tagged subunit polypeptides; 2) Generation and production of monoclonal antibodies directed against mapped epitopes of target subunits; and 3) Development of small- and large-scale antibody affinity purification for multi-subunit complexes. Indeed, each of the 3 projects proposed in this program will require the large-scale production and purification of molecular machines. These represent the principal protein complexes targeted for functional and structural studies using a combination of cryo-EM/single particle reconstruction, X-ray crystallography, and single molecule FRET analysis. In each of the 3 projects, overlapping sets of proteins and protein complexes such as human TFIID, CRSP, ARC, PBAF, RNA polymerase II, the pre-initiation complex (PIC), and their attendant sub-complexes, as well as activator binding factors, will be the primary subjects of study. In most cases, these different multi- subunit assemblages will be isolated from human cells, but in some instances, depending on the exact nature of the experimental strategy, we will also make use of analogous functional complexes isolated from Drosophila. Regardless of the original cell type or species of the starting material, a common set of experimental strategies and related protocols will need to be developed in order to carry out the structural studies that form the scientific basis of this program project.

开发该计划项目的主要动力是巩固和协调我们的努力建立一个核心实验室，旨在促进和纯化大型多元与基因转录有关的亚基调节复合物。因此，建立高效，整合和相互依存的核心设施，以满足这些相当大实验挑战对于该计划项目的成功至关重要。因此，我们有将核心实验室细分为3个不同的分区，每个分区都有其自身定义的特定目的集： 1）表达系统的构建和标记的亚基多肽的生产； 2）针对映射表位的单克隆抗体的产生和生产目标亚基； 3）开发小型和大型抗体亲和力纯化多支化合物复合物。确实，该计划中提出的三个项目中的每个项目都将需要大规模生产和分子机器的纯化。这些代表了针对的主要蛋白质复合物用于使用冷冻EM/单个粒子重建的功能和结构研究， X射线晶体学和单分子FRET分析。在这三个项目中的每个项目中，重叠蛋白质和蛋白质复合物（例如人TFIID，CRSP，ARC，PBAF，RNA）的集合集聚合酶II，启动前复合物（PIC）及其随之而来的子复合物以及激活因子的结合因子将是研究的主要主题。在大多数情况下，这些不同的多亚基组合将从人类细胞中分离出来，但在某些情况下，取决于实验策略的确切性质，我们还将利用类似的功能从果蝇分离的复合物。不论起始的原始细胞类型或种类材料，一组常见的实验策略和相关协议，需要制定为了进行构成该计划项目的科学基础的结构研究。