Protein Production, Analysis and Assay Development

蛋白质生产、分析和检测开发

• 批准号: 7434199
• 负责人: John H Elder
• 金额: $ 31.57万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-18 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7434199
• 关键词: Cells; Collaborations; Development; Drug resistance; Endopeptidases; Escherichia coli; Feline Immunodeficiency Virus; Gagging; HIV-1; HIV-1 protease; Literature; Patients; Peptide Hydrolases; Production; Proteins; Purpose; Reagent; Recombinants; Relative (related person); Reporting; Role; SIV; Sampling; Side; Site; Specificity; System; Testing; Toxic effect; Variant; assay development; base; biosafety level 3 facility; enzyme substrate; inhibitor/antagonist; mutant; novel strategies; pol Gene Products; pol genes; polypeptide; programs; protein expression; tissue culture

The purpose of the Core is to prepare, purify, and test all recombinant, wild type and drug resistant proteases and prepare polypeptide and fluorigenic substrates required for the overall Program Project. This Core will provide the reagents and background testing required of all the projects, including studies of substrate/inhibitor specificity (Olson, Torbett), inhibitor development (Finn/Sharpless, Stout/McCree) and structural analyses of drug-resistant proteases (Stout Core/Torbett). Additionally, the Protein Expression Core will interact directly with the other Cores to characterize predominant drug resistant proteases revealed in analyses of patient samples (Looney) and provide enzymes and substrates required for crystallographic analyses (Stout). In the previous years of this ongoing and productive collaboration, we have prepared and purified over 50 wild type, drug-resistant, and mutant proteases from HIV-1, FIV, and SIV, each cloned and over-expressed in E. coli. These constructs will be maintained for use by the program and the repertoire will be expanded as more sequences become available. Highly purified proteases will be prepared and supplied as detailed below. In addition, new proteases based on structural changes associated with drug resistance that are identified by the projects will be prepared for analysis of relative Kcat and Km values as well as to assess the relative Ki of various inhibitors in the context of these proteases. The Core will also continue to develop additional substrates from regions of the Gag-Pol polyprotein, including the primary cleavage sites on either side of NC as well as seven other cleavage sites in Gag-Pol. Where applicable, ex vivo tissue culture analyses of inhibitor efficacy and specificity will be carried out, primarily using a pseudovirion expression system developed by this Core that allows assessment of efficacy of PR cleavage of Gag and Gag/Pol polyproteins, as well as inhibitor potency, specificity and cell toxicity. Tests of efficacy against infectious HIV-1 will be performed in BSL-3 facilities under the direction of the Torbett component.

核心的目的是制备、纯化和测试所有重组、野生型和抗药性 并制备整个计划项目所需的多肽和生氟底物。这 CORE将提供所有项目所需的试剂和背景测试，包括研究 底物/抑制剂专一性(Olson，Torbett)，抑制剂开发(Finn/Sharpless，Stout/McCree)和 耐药蛋白酶的结构分析(Stout Core/Torbett)。此外，蛋白质的表达 CORE将直接与其他核心相互作用，以表征所揭示的主要耐药蛋白 在病人样本分析中(Looney)，并提供结晶学所需的酶和底物 分析(Stout)。在这一持续和富有成效的合作的前几年中，我们准备并 从HIV-1、FIV和SIV中纯化了50多种野生型、抗药性和突变的蛋白酶，每个酶都被克隆和 在大肠杆菌中过度表达。这些构造将被维护以供程序使用，并且曲目将 随着更多的序列变得可用而被扩展。将制备和供应高纯度的蛋白酶 具体如下。此外，基于结构变化的新蛋白水解酶与耐药性有关 将为分析项目确定的相对Kcat和Km值以及 在这些蛋白酶的背景下，评估不同抑制剂的相对KI。核心也将继续 从Gag-Pol多蛋白的区域产生额外的底物，包括主要的切割位点 在NC的两侧以及Gag-Pol的其他七个裂解位点上。适用的情况下，体外组织 将主要使用假病毒粒子对抑制物的有效性和特异性进行培养分析。 由该核心开发的表达系统，可以评估GAG和PR切割的有效性 GAG/POL多蛋白，以及抑制剂的效力、特异性和细胞毒性。对药物的效力测试 在Torbett部门的指导下，将在BSL-3设施中进行传染性艾滋病毒-1检测。