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Protein Expression and Libraries Facility

蛋白质表达和文库设施

基本信息

批准号：
7945026
负责人：
PAUL M LIEBERMAN
金额：
$ 16.85万
依托单位：
WISTAR INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-04-15 至 2014-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7945026
关键词：
Accounting Affinity Aliquot Antibiotic Resistance Antibodies Antibody Formation Archives Area Arts Bacteria Baculoviruses Basic Cancer Research Biological Assay Biological Process Buffers Cancer Center Cancer Research Project Cell Culture Techniques Cell Line Cell Surface Proteins Cell Wall Cells Characteristics Chimeric Proteins Chromatography Cleaved cell Cloning Cloning Vectors Collection Complementary DNA Consult Consultations Crystallization Culture Media Custom Cytomegalovirus DNA Restriction Enzymes DNA Sequence Analysis DNA-Directed RNA Polymerase Derivation procedure Destinations Detection Detergents Development Dialysis procedure Digestion Drug resistance Engineering Ensure Epitopes Escherichia coli Expression Library Factor X Faculty Fluorescence Microscopy Freezing Gene Delivery Gene Expression Generations Genes Genetic Genetic Variation Genomic Library Glutathione S-Transferase Goals Gold Growth Guidelines Healthcare Histidine Human Resources Hybridomas Immunization In Vitro Inclusion Bodies Incubators Individual Infection Insecta Isopropyl Thiogalactoside Kinetics Libraries Life Maintenance Mammalian Cell Membrane Methodology Microscopy Molecular Sieve Chromatography Monitor Mus N-terminal Nucleic Acids Peptide Hydrolases Peptide Signal Sequences Performance Plasmids Preparation Procedures Process Production Protein Secretion Proteins Protocols documentation Quality Control RNA Interference Recombinant DNA Recombinant Proteins Recombinants Research Personnel Resources Retroviral Vector Retroviridae Role Sampling Scientist Screening procedure Sepharose Services Site-Directed Mutagenesis Sonication Source Standardization Structure Structure-Activity Relationship Subfamily lentivirinae System Technology Temperature Tetracyclines Thrombin Training Transfection Transfer RNA Ubiquitin C Viral Virion Virus Western Blotting Yeasts aerosolized anticancer research assay development base cancer stem cell cell type experience expression vector gel electrophoresis high throughput screening improved in vivo instrument interest loss of function meetings member particle plasmid DNA polypeptide prevent promoter protein expression protein folding protein purification quality assurance receptor recombinant virus repository research study small hairpin RNA small molecule small molecule libraries stable cell line stem cell population technology development tissue culture trioctyl phosphine oxide vector

项目摘要

The Protein Expression and Libraries Facility (Protein Expression) provides Cancer Center members efficient and expert technical assistance in recombinant DMA plasmid engineering, protein expression in bacteria and baculovirus-infected insect cells, purification of recombinant proteins to homogeneity, and production of high-titer stocks of retroviruses (e.g. lentiviruses) for delivery of shRNA and cDNAs to mammalian cells. The Facility has expertise in all aspects of vector technology for protein expression, baculovirus generation, recombinant protein expression, and affinity and conventional chromatography approaches to protein purification, and production of infectious lenti-/retroviruses. The proteins produced by the facility must be of high quality and purity in order to be used by Cancer Center scientists to achieve a wide range of experimental objectives, such as characterization of enzymatic activities, crystallization for structural analysis, characterization of structure-function relationships of protein:protein, protein:nucleic acid, and protein:small molecule interactions; development of assays for small molecule high throughput screening; and immunization of mice to generate custom antibodies utilizing the Hybridoma Facility. The facility will continue to expand it's repertoire of vectors available for protein expression, included high titer retroviral vectors, acquire RNAi libraries, and implement a new assay development service for highthroughput screening of small molecule libraries. The facility personnel are highly trained technical experts in all areas of the proposed support services to be provided to the individual projects. The centralization and standardization of these practices under the direction of very experienced staff allows for high-throughput expression plasmid construction and large-volume protein expression services, including quality assurance and control procedures to ensure efficient, consistent production and purification of high quality recombinant proteins. The Facility also maximizes biosafety by confining retrovirus (e.g. lentivirus) production to a centralized biosafety level 2 (BSL2) unit, which prevents aerosolization of viruses from contaminating incubators and parental cultures of cell lines, and improves quality control in the production of virus stocks to be used in gain- and loss-of-function experiments in vitro and in vivo. Currently the Facility is being expanded to accommodate the storage and handling of viral libraries, additional purification instruments, and the rapid assay systems to meet Cancer Center needs.

蛋白质表达和文库设施（蛋白质表达）为癌症中心成员提供在重组 DMA 质粒工程、蛋白质表达方面提供高效、专业的技术援助细菌和杆状病毒感染的昆虫细胞，纯化重组蛋白至均质，以及生产高滴度逆转录病毒（例如慢病毒）库存，用于将 shRNA 和 cDNA 递送至哺乳动物细胞。该设施拥有蛋白质表达载体技术各个方面的专业知识，杆状病毒生成、重组蛋白表达、亲和力和常规层析蛋白质纯化方法以及感染性慢病毒/逆转录病毒的生产。所产生的蛋白质该设施必须具有高质量和纯度，以便癌症中心的科学家能够使用它来实现广泛的实验目标，例如酶活性的表征、结晶结构分析，蛋白质：蛋白质，蛋白质：核酸的结构功能关系表征，和蛋白质：小分子相互作用；小分子高通量检测方法的开发筛查；利用杂交瘤设施对小鼠进行免疫以产生定制抗体。这该设施将继续扩大可用于蛋白质表达的载体库，包括高效价逆转录病毒载体，获取 RNAi 文库，并实施新的高通量检测开发服务小分子文库的筛选。设施人员都是训练有素的技术专家拟议向各个项目提供支持服务的所有领域。集中化和在经验丰富的员工的指导下，这些实践的标准化可以实现高通量表达质粒构建和大容量蛋白质表达服务，包括质量保证和控制程序，以确保高效、一致的生产和纯化高质量的重组体蛋白质。该设施还通过将逆转录病毒（例如慢病毒）生产限制在一定范围内，最大限度地提高生物安全性。集中式生物安全 2 级 (BSL2) 装置，可防止病毒雾化污染细胞系的培养箱和亲代培养物，并改进病毒库存生产的质量控制，以可用于体外和体内功能获得和丧失的实验。目前该设施正在扩展以容纳病毒库的存储和处理、额外的纯化仪器，以及满足癌症中心需求的快速检测系统。