Protein Expression and Libraries Facility

蛋白质表达和文库设施(蛋白质表达)为癌症中心成员提供 高效和专业的重组DMA质粒工程技术援助，蛋白质在 细菌和杆状病毒感染的昆虫细胞，重组蛋白的纯化，以同质，以及 生产高滴度逆转录病毒(如慢病毒)，用于将shRNA和cDNA输送到 哺乳动物细胞。该设施在蛋白质表达载体技术的各个方面都有专业知识， 杆状病毒的产生、重组蛋白的表达、亲和层析和常规层析 蛋白质提纯和传染性兰蒂/逆转录病毒的生产方法。产生的蛋白质是由 该设施必须是高质量和高纯度的，才能被癌症中心的科学家用来实现 广泛的实验目标，如表征酶的活性，结晶 结构分析，表征蛋白质：蛋白质，蛋白质：核酸， 和蛋白质：小分子相互作用；小分子高通量分析的发展 筛选；以及利用杂交瘤设施对小鼠进行免疫以产生定制抗体。这个 设施将继续扩大其可用于蛋白质表达的载体库，包括高滴度 逆转录病毒载体，获取RNAi文库，并实施新的高通量测试开发服务 小分子文库的筛选。该设施的工作人员是训练有素的技术专家 拟向个别项目提供的所有领域的支助服务。中央集权和 在非常有经验的员工的指导下对这些实践进行标准化，从而实现高吞吐量 表达载体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、一致的生产和高质量重组人的纯化 蛋白质。该设施还通过将逆转录病毒(如慢病毒)的生产限制在 集中式生物安全二级(BSL2)单元，可防止病毒的气雾化污染 细胞系的孵化器和亲本培养，并改进生产病毒库的质量控制，以 可用于体外和体内的功能获得和丧失实验。目前，该设施正在 扩展以容纳存储和处理病毒库、额外的纯化仪器以及 满足癌症中心需求的快速检测系统。

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.