Protein Core

核心能力和方法：蛋白质核心积累并展示了利用细菌和昆虫细胞系统进行重组蛋白质表达和纯化所需的专业知识。蛋白质表达平台将以6X-His和/或Strep标记的蛋白质为中心，有或没有其他融合伙伴，如GB1、硫氧还蛋白、NusA、MBP和GST。融合构建体将包含TEV或凝血酶裂解位点，用于下游去除融合蛋白。对于每个表达载体变体，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。对于在大肠杆菌中的生产，优化方法将包括：1)编码序列的增量截断/延伸以定义最小折叠结构域，2)在保存的生物功能的背景下编码序列的突变或体外进化，以及3)和2)的组合。这种方法将导致大量的构建，需要高通量的克隆、表达和溶解性筛选。核心已经成功地处理了这些任务，并以结构确定的形式产生了困难的蛋白质，如VPR和VPX。尽管做出了这些努力，但可溶物质的生产 大肠杆菌中的病毒来源或细胞蛋白可能无法达到某些目标。在这些情况下，靶标将在昆虫细胞中表达。已建立了高通量的真核系统克隆、表达筛选和蛋白质生产方法，并用于TRIMSA、DCAFi和 DDBi。纯化的蛋白质将通过静态多角度光散射来评估其季态，通过LC-ESI-TOF质谱仪来确认纯度，核磁共振同位素标记效率和硒蛋氨酸(Se-Met)标记，然后提交样品进行核磁共振和X射线结晶学。标签协议)是公认的并经常使用的。用于从真核表达中纯化的蛋白质 系统，翻译后修饰将使用电喷雾飞行时间和其他质谱学方法进行分析。为了确定蛋白质的稳定性，将进行圆二色谱、荧光光谱和差示扫描量热法作为温度和杂色剂的函数。 三、项目组成部分：蛋白质核心一直是PCHPI对VPR和DCAFi48的研究的主要驱动力)，并将继续在这些研究中发挥主要作用。

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).