Core--Protein expression and purification

核心--蛋白表达与纯化

• 批准号: 6565130
• 负责人: Steven T. Olson
• 金额: $ 21.47万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6565130
• 关键词: biomedical facility; biotechnology; cell line; gene expression; protease inhibitor; protein purification; recombinant proteins; serine proteinases

The protein expression and purification core will provide purified recombinant serpins and other needed proteins from both bacterial and mammalian cell sources to all four research projects. Using bacterial expression plasmids or stably-transfected BHK cell lines, both constructed by the Molecular Biology and Cell Culture core or already available in the laboratories of the investigators, the core will express the required protein by growth of the appropriate cell type, harvest either bacteria or BHK cell growth medium, and isolate the recombinant protein to the high degree of purity required by each project. The core will use established purification methods developed by the investigators for proteins already expressed, but will further optimize protocols to maximize yield and streamline purification. Development for protocols for isolation of new proteins will be the responsibility of the technical director of the core in consultation with the appropriate P.I. The core will provide quality control of the purified protein, by electrophoretic means, functional assay, and where appropriate protein sequencing. The Administrative Director will schedule protein purifications to meet the priorities of the investigators, but with the aim of providing equitable service to all projects. The core may expand beyond the present personnel to meet additional protein needs of the four investigators for other funded projects. However, such additional services will be charged to the other grants.

蛋白表达和纯化核心将为所有四个研究项目提供纯化的重组丝氨酸蛋白酶抑制剂和其他所需的蛋白质，这些蛋白质来自细菌和哺乳动物细胞来源。使用细菌表达质粒或稳定转染的BHK细胞系（均由分子生物学和细胞培养核心构建或已在研究者实验室中获得），核心将通过适当细胞类型的生长表达所需蛋白质，收获细菌或BHK细胞生长培养基，并分离重组蛋白质至每个项目所需的高纯度。核心将使用研究人员为已经表达的蛋白质开发的已建立的纯化方法，但将进一步优化方案，以最大限度地提高产量和简化纯化。新蛋白质分离方案的制定将由核心技术总监与适当的P.I.协商后负责。核心将通过电泳方法、功能测定和适当的蛋白质测序提供纯化蛋白质的质量控制。行政主任将安排蛋白质纯化工作，以满足调查人员的优先事项，但目的是为所有项目提供公平的服务。核心人员可能会扩大到现有人员以外，以满足其他资助项目的四名调查员对蛋白质的额外需求。不过，这些额外服务将由其他赠款支付。