Protein Core

蛋白质核心

• 批准号: 8899577
• 负责人: jinwoo ahn
• 金额: $ 41.52万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8899577
• 关键词: Biological Process; Cells; Chimeric Proteins; Circular Dichroism; Code; Complex; Core Protein; Differential Scanning Calorimetry; Escherichia coli; Evolution; Excision; Fluorescence Spectroscopy; Funding; HIV; Human; In Vitro; Insecta; Integration Host Factors; Isotope Labeling; Label; Mass Spectrum Analysis; Methodology; Methods; Mission; Mutagenesis; Play; Post-Translational Protein Processing; Production; Protein Family; Proteins; Protocols documentation; Reagent; Recombinant Proteins; Role; Sampling; Selenomethionine; Services; Site; Solubility; Stream; Structure; System; Temperature; Testing; Thioredoxin; Thrombin; Time; Variant; Virus; X-Ray Crystallography; driving force; expression cloning; expression vector; light scattering; mutant; protein complex; protein expression; protein purification; screening; tool; vpr Gene Products

Core Capabilities & Approach: The Protein Core has accumulated and demonstrated the required expertise in recombinant protein expression and purification utilizing bacterial and insect cell systems. The protein expression platform will be centered on 6X-His and/or Strep tagged proteins, with or without other fusion partners, such as GB1, thioredoxin, NusA, MBP, and GST. Fusion constructs will contain TEV or thrombin cleavage sites for down-stream removal of the fusion protein. For each expression vector variant, multiple conditions will be tested to achieve optimal expression and yield: host, temperature, induction time, etc. For production in E. coli, the optimization approaches will include: 1) incremental truncation/extension of the coding sequence to define minimal folding domains, 2) mutagenesis or in vitro evolution of the coding sequence in the context of preserved biological function, and 3) a combination of) and 2). This approach will result in large numbers of constructs, requiring high-throughput cloning, expression, and solubility screening. These tasks have been successfully handled by the Core and yielded difficult proteins such as Vpr and Vpx in forms amenable to structure determination. Despite these efforts, production of soluble virus-derived or cellular proteins in E. coli may fail for some targets. In these cases, targets will be expressed in insect cells. The high-throughput methodologies for cloning, expression screening, and protein production in eukaryotic systems have been established and utilized for expression and purification of Trimsa, DCAFi, and DDBi. Purified proteins will be characterized by static multi-angle light scattering to assess their quaternary state, LC-ESI-TOF mass spectrometry to confirm purity, NMR isotope labeling efficiency and selenomethionine (Se-Met) labeling, before submitting samples for NMR and X-ray crystallography. Labeling protocolsf^39-242) are well-established and routinely used. For proteins purified from eukaryotic expression systems, post-translational modification will be analyzed using ESI-TOF and other mass spectrometry methods. To determine protein stability, circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry will be performed as a function of temperature and chaotropic agents. iii. Project Component: The Protein Core has been the main driving force in the PCHPI's studies of Vpr and DCAFif48,86) and will continue to play a primary role in these studies (Ps).

核心能力和方法：蛋白质核心已经积累并证明了利用细菌和昆虫细胞系统进行重组蛋白表达和纯化所需的专业知识。蛋白质表达平台将集中在6X-His和/或Strep标记的蛋白质上，有或没有其他融合伴侣，如GB 1、硫氧还蛋白、NusA、MBP和GST。融合构建体将含有TEV或凝血酶切割位点，用于融合蛋白的下游去除。对于每种表达载体变体，将测试多种条件以实现最佳表达和产量：宿主、温度、诱导时间等。在大肠杆菌中，优化方法将包括：1）编码序列的增量截短/延伸以限定最小折叠结构域，2）在保留生物学功能的情况下编码序列的诱变或体外进化，和3））和2）的组合。这种方法将产生大量的构建体，需要高通量克隆、表达和溶解度筛选。这些任务已成功地处理的核心，并产生了困难的蛋白质，如Vpr和Vpx的形式服从结构测定。尽管有这些努力，可溶性的 E.大肠杆菌可能对某些靶标无效。在这些情况下，靶标将在昆虫细胞中表达。已经建立了用于真核系统中的克隆、表达筛选和蛋白质生产的高通量方法，并用于表达和纯化Trimsa、DCAFi和DCAFi。 DDBi。在提交样品进行NMR和X射线晶体学分析之前，将通过静态多角度光散射对纯化的蛋白质进行表征，以评估其四级状态，LC-ESI-TOF质谱法确认纯度，NMR同位素标记效率和硒代蛋氨酸（Se-Met）标记。标记方案^[39 -242]已经得到了很好的建立，并被常规使用。对于从真核表达纯化的蛋白质 系统，翻译后修饰将使用ESI-TOF和其他质谱方法进行分析。为了确定蛋白质的稳定性，圆二色性、荧光光谱和差示扫描量热法将作为温度和离液剂的函数进行。 三.项目组成部分：蛋白质核心一直是PCHPI研究Vpr和DCAFif 48，86）的主要驱动力，并将继续在这些研究中发挥主要作用（Ps）。