Protein Core

蛋白质核心

基本信息

批准号：
8528175
负责人：
jinwoo ahn
金额：
$ 45.7万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8528175
关键词：
Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly 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 Screening procedure 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 tool vpr Gene Products

项目摘要

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的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6倍-HIS和/或链球菌标记的蛋白质上，有或没有其他融合伴侣，例如GB1，Thioredoxin，NUSA，MBP和GST。融合构建体将包含TEV或凝血酶裂解位点，以下游去除融合蛋白。对于每个表达矢量变体，将测试多种条件以实现最佳表达和产量：宿主，温度，诱导时间等用于大肠杆菌的生产，优化方法将包括：1）编码顺序的增量截断/扩展，以定义最小折叠域，2）2）杂物化或在序列中的代码序列和3次）和3）的编码序列和3次。这种方法将导致大量构造，需要高通量克隆，表达和溶解度筛选。这些任务已被核心成功处理，并产生了难以符合结构确定的形式的困难蛋白质。尽管做出了这些努力，但生产可溶性大肠杆菌中的病毒衍生或细胞蛋白可能会失败某些靶标。在这些情况下，靶标将在昆虫细胞中表达。已经建立并利用了真核系统中克隆，表达筛选和蛋白质产生的高通量方法，用于表达和纯化Trimsa，DCAFI和 DDBI。纯化的蛋白质将以静态多角度光散射为特征，以评估其Quaternary LC-ESI-TOF质谱法，以确认纯度，NMR同位素标记效率和硒硫氨酸（SE-MET）标记，然后在提交NMR和X射线晶体学的样品之前。标记协议F^39-242）已建立良好且常规使用。从真核表达纯化的蛋白质将使用ESI-TOF和其他质谱法分析系统，翻译后修饰。为了确定蛋白质稳定性，将作为温度和交际剂的函数进行圆形二色性，荧光光谱和差异扫描量热法。 iii。项目组成部分：蛋白质核心一直是PCHPI对VPR和DCAFIF48,86的研究的主要驱动力，并且将在这些研究中继续发挥主要作用（PS）。