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蛋白和其他所需蛋白质。使用细菌表达质粒或稳定转染的BHK细胞系，无论是由分子生物学和细胞培养核心构建的，还是在研究人员的实验室中已有的，核心将通过生长适当的细胞类型来表达所需的蛋白质，收获细菌或BHK细胞生长培养基，并将重组蛋白分离到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。新蛋白分离方案的开发将由核心技术总监负责，并与相应的pi协商。核心将通过电泳手段、功能测定和适当的蛋白质测序对纯化蛋白进行质量控制。行政主任将安排蛋白质纯化工作，以满足研究人员的优先事项，但目的是为所有项目提供公平的服务。核心人员可能会在现有人员之外扩大，以满足其他资助项目的四名研究人员的额外蛋白质需求。但是，这些额外服务将从其他赠款中支付。

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.