Protein Design, Expression and Purification Core

蛋白质设计、表达和纯化核心

• 批准号: 8931201
• 负责人: Maria Schumacher
• 金额: $ 11.31万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-25 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8931201
• 关键词: Affinity; Animal Model; Antifungal Agents; Applications Grants; Baculoviruses; Binding Sites; Biochemical; Calcineurin; Codon Nucleotides; Complex; Crystallization; Development; Ensure; Escherichia coli; Excision; Feedback; Gene Proteins; Generations; Genes; Genomic DNA; Goals; Hand; Human; Label; Methodology; Modification; Molecular; Monitor; Mutagenesis; Mutation; Outcome; Output; Pathology; Phase; Plant Resins; Plasmids; Procedures; Production; Protein Binding; Protein Overexpression; Proteins; Reagent; Research Personnel; Resolution; Scientist; Selenomethionine; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Site; Solubility; Structural Protein; Structure; System; Temperature; Testing; The science of Mycology; Time; Work; cost effectiveness; design; design and construction; experience; fungus; gene cloning; in vivo; inhibitor/antagonist; member; mutant; novel; overexpression; programs; protein complex; protein expression; protein purification; structural biology; success; vector

ABSTRACT: Protein Design, Expression, and Purification Core This proposal represents a unique academic effort that weds structural biology, molecular mycology, and vertebrate pathology to target the signal transduction pathways that allow pathogenic fungi to survive in humans. The structural approaches, which are essential to the success of the proposed Aims, will require the production of large quantities of pure and homogeneous proteins. Many of these proteins or protein complexes will be difficult to express and those that do express may also be difficult to produce in a soluble form. Thus, it is likely that multiple expression constructs will need to be tested for production of these soluble and pure target proteins. The Specific Aims of this grant application are much better served if these activities are centralized as cost effectiveness is greatly enhanced by the removal of redundant efforts and the ability to bring to all projects technical scientists whose sole purpose is to maximize the output of biochemically active proteins. Thus, there are three general aims of goals of the Protein Design, Expression and Purification Core (PDEP): Specific Aim 1 Generation of multiple expression constructs for target proteins and complexes. Genomic DNA, or artificially produced genes, will be inserted into multiple commercially available vectors providing a high likelihood of success. Specific Aim 2 Testing for target constructs protein expression levels and solubility. Once cloned the PDEP will optimize the overexpression of soluble proteins using a 96 well plate format and multiple expression systems. Specific Aim 3 Testing and optimizing expression constructs and performing final purification. The PDEP will purify proteins through affinity resins and help isotopically label proteins for NMR studies. The proteins will be distributed to core members for biochemical and structural studies. The most crucial goals of these studies will be high-resolution structure determination and the subsequent design of specific inhibitors.

摘要：蛋白质设计、表达和纯化核心 该提案代表了一项独特的学术努力，它将结构生物学、分子真菌学和 脊椎动物病理学以信号转导途径为目标，使病原真菌能够在 人类。结构性方法对于拟议目标的成功至关重要，需要 生产大量纯且均质的蛋白质。许多这些蛋白质或蛋白质 复合物将难以表达，并且表达的复合物也可能难以以可溶性形式产生 形式。因此，很可能需要测试多个表达构建体来生产这些可溶性的 和纯的目标蛋白。如果这些活动能够更好地实现本次赠款申请的具体目标 集中化，因为通过消除多余的工作和能力大大提高了成本效益 为所有项目带来技术科学家，其唯一目的是最大限度地提高生化活性物质的产量 蛋白质。因此，蛋白质设计、表达和纯化核心的目标有三个总体目标 (PDEP)：具体目标 1 生成靶蛋白和复合物的多种表达构建体。 基因组DNA或人工产生的基因将被插入到多个市售载体中 提供很高的成功可能性。具体目标 2 测试目标构建体蛋白表达水平 和溶解度。一旦克隆，PDEP 将使用 96 孔板优化可溶性蛋白的过度表达 格式和多种表达系统。具体目标 3 测试和优化表达构建体 进行最终纯化。 PDEP 将通过亲和树脂纯化蛋白质并帮助同位素标记 用于 NMR 研究的蛋白质。这些蛋白质将分配给核心成员进行生化和结构分析 研究。这些研究最重要的目标是高分辨率结构测定和 后续设计特异性抑制剂。