Protein Core

核心能力和方法：Protein Core已经积累并展示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。该蛋白表达平台将以6X-His和/或链球菌标记蛋白为中心，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，用于融合蛋白的下游去除。对于每个表达载体变体，将测试多种条件以达到最佳表达和产量：宿主、温度、诱导时间等。对于大肠杆菌的生产，优化方法将包括：1)编码序列的增量截断/扩展以定义最小折叠结构域，2)在保留生物功能的背景下对编码序列进行诱变或体外进化，以及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。Core已经成功地处理了这些任务，并以适合结构测定的形式产生了Vpr和Vpx等困难的蛋白质。尽管这些努力，生产可溶性

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).