Protein Design, Expression and Purification Core

摘要：蛋白质的设计、表达和纯化核心 这一建议代表了一项独特的学术努力，将结构生物学、分子真菌学和 脊椎动物病理学以信号转导通路为靶点，使病原真菌在 人类。结构性方法对于拟议目标的成功至关重要，它将需要 生产大量纯净、均一的蛋白质。这些蛋白质或蛋白质中的许多 复合体将很难表达，而那些确实表达的复合体也可能很难在可溶性中生产 形式。因此，可能需要测试多个表达构建体以生产这些可溶的 和纯净的目标蛋白。如果这些活动能更好地服务于这项赠款申请的具体目标 是集中的，因为成本效益通过消除多余的工作和以下能力而大大增强 将唯一目的是最大化生物化学活性产出的技术科学家带到所有项目中来 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)：特定目标1为目标蛋白和复合体生成多种表达构建体。 基因组DNA，或人工产生的基因，将被插入到多种商业可用载体中 提供了很高的成功可能性。特定目标2检测目标构建体蛋白质表达水平 和溶解度。一旦被克隆，PDEP将利用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测试和优化表达结构和 正在进行最终提纯。PDEP将通过亲和树脂纯化蛋白质，并帮助同位素标记 用于核磁共振研究的蛋白质。这些蛋白质将分配给核心成员进行生化和结构分析 学习。这些研究的最关键目标将是高分辨率结构确定和 随后设计特定的抑制剂。

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.