Protein Expression and Proteomics

蛋白质表达和蛋白质组学（PEP）核心获得了为期一年的补充资金， 2006年6月1日，为我们的调查人员社区提供未满足的需求。蛋白质表达部分 在约翰·埃尔德博士的指导下，拟议的试点核心将提供适当的载体， 细胞系，以促进表达克隆的靶向蛋白质。蛋白质组学部分 在布鲁斯托贝特博士的指导下，该中心将为CFAR提供蛋白质分析服务 investigators.这些服务包括从病毒、细胞和组织中提取蛋白质; 使用一维和二维（D）电泳和成像系统进行定量;蛋白质 使用质谱进行鉴定;使用Luminex多路复用进行蛋白质分析 检测技术PEP核心的独特之处在于专注于为调查人员提供服务 需要表达来自HIV-1和其它病毒基因的蛋白质以及HIV-1和其它病毒基因的蛋白质分析 病毒感染的样本 核心的蛋白表达部分将提供所需的适当载体和细胞系， 使用细菌、昆虫细胞或哺乳动物细胞，促进表达克隆靶向突变体的蛋白质 载体系统，如认为适合于特定应用。埃尔德实验室编制了一份 为每种表达系统提供广泛的载体组，并在蛋白质表达方面具有丰富的经验 和纯化用于配体结合研究、酶学和晶体学分析。实验室是 配备有在所需温度下培养细胞所需的培养箱以及离心机，FPLC， 和HPLC系统以在必要时帮助蛋白质纯化。核心将协助调查员 载体选择和靶基因的扩增和克隆。此外，核心将帮助调查人员 在优化蛋白质表达和选择最适合于每种蛋白质的纯化方案方面， 应用程序. 核心的蛋白质组学部分将提供从可视化到 识别. CFAR成员将有机会获得NCI资助的蛋白质组学核心， 斯克里普斯研究所分子和实验医学系。目前 Proteomics Core提供1-D和2-D凝胶电泳蛋白质分离仪器，IEF 用于溶液相IEF的变焦分级器，这是一种最先进的2D蛋白质组成像系统，配有Bio-Rad VersaDoc 1000相机和成像软件。所有成像软件都可以免费使用， 核心内的常驻计算机核心技术人员可提供技术建议， 必要时，核心技术人员提供分析和成像软件的演示和研讨会。在 此外，核心技术人员将为研究人员提供优化蛋白质提取程序的建议 以及选择最适合每种应用的纯化方案。 蛋白质表达和蛋白质组学核心将提供有关核心的信息研讨会/会议 服务，并在整个资助期内举办“新科技”研讨会/讲座。

The Protein Expression and Proteomics (PEP) Core received supplemental funding for one year starting June 1, 2006, to provide an unmet need for our community of investigators. The Protein Expression portion of the proposed pilot Core, under the direction of Dr. John Elder, will provide the appropriate vectors and cell lines needed to facilitate expression cloning of investigator-targeted proteins. The Proteomics portion of the core, under the direction of Dr. Bruce Torbett, will provide protein analysis services for CFAR investigators. These services include protein extraction from viruses, cells, and tissues; protein resolution and quantification using 1- and 2-dimensional (D) electrophoresis and imaging systems; protein identification using mass spectroscopy; and analyses of proteins using Luminex¿ xMAP¿ multiplex detection technology. The unique aspect of the PEP Core is the focus on providing services to investigators requiring expression of proteins from HIV-1 and other viral genes and protein analysis of HIV-1 and other virally infected samples. The Protein Expression portion of the Core will provide the appropriate vectors and cell lines needed to facilitate expression cloning of investigator-targeted proteins, using bacterial, insect cell, or mammalian cell vector systems, as deemed appropriate for the particular application. The Elder laboratory has compiled an extensive panel of vectors for each expression system and has extensive experience in protein expression and purification for ligand binding studies, enzymology, and crystallographic analyses. The laboratory is equipped with incubators required to grow cells at the required temperatures and also centrifuges, FPLC, and HPLC systems to aid in protein purification where necessary. The Core will aid the investigator in vector selection and amplification and cloning of targeted genes. In addition, the Core will help investigators in optimizing protein expression and selection of purification schemes most appropriate for each application. The Proteomics portion of the Core will provide all facets of protein resolution from visualization to identification. CFAR members will have access to the NCI-funded Proteomics Core housed within the Departmental of Molecular and Experimental Medicine at The Scripps Research Institute. Currently the Proteomics Core has available 1- and 2-D gel electrophoresis protein separation instrumentation, IEF Zoom fractionators for solution phase IEF, a state-of-the-art 2D Proteomic Imaging System with a Bio-Rad VersaDoc 1000 imager and imaging software. All imaging software can be utilized free of charge on resident computers housed within the Core. Technical advice is available from the Core technicians, and when needed, Core technicians provide demonstrations and seminars on analysis and imaging software. In addition, Core technicians will provide advice to investigators for optimizing protein extraction procedures and selection of purification schemes most appropriate for each application. The Protein Expression and Proteomics Core will provide informational seminars/sessions on the Core services each year and "new technology" seminars/sessions throughout the funding period.