Core--Protein expression and purification

蛋白质表达和纯化核心将为所有四个研究项目提供来自细菌和哺乳动物细胞来源的纯化重组蛇毒和其他所需蛋白质。使用由分子生物学和细胞培养核心构建的或研究人员实验室已有的细菌表达载体或稳定转染的BHK细胞系，核心将通过生长适当的细胞类型来表达所需的蛋白质，收集细菌或BHK细胞生长介质，并分离重组蛋白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既定纯化方法，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主管负责，并与适当的P.I.协商。核心将通过电泳手段、功能分析和适当的蛋白质测序对纯化的蛋白质进行质量控制。行政主任将安排蛋白质纯化，以满足调查人员的优先事项，但目的是向所有项目提供公平的服务。核心可能会扩大到现有人员之外，以满足四名研究人员对其他资助项目的额外蛋白质需求。然而，这些额外的服务将从其他补助金中收取。

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.