Protein Production in the Medical Structural Genomics of Pathogenic Protozoa

致病性原生动物医学结构基因组学中的蛋白质生产

• 批准号: 7071313
• 负责人: WESLEY C VAN VOORHIS
• 金额: $ 50.2万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7071313
• 关键词: Escherichia coli; X ray crystallography; antiprotozoal agents; bioassay; bioinformatics; cell free system; crystallization; drug discovery /isolation; evaluation /testing; gene expression; high throughput technology; method development; mixed tissue /cell culture; peptide chemical synthesis; protein folding; protein purification; protein quantitation /detection; protein sequence; protein structure function; proteomics; robotics; site directed mutagenesis; structural biology; transfection /expression vector

The Medical Structural Genomics of Pathogenic Protozoa (MSGPP) has the objective of producing ligand-structure information for drug development from 10 protozoa of medical and bioterrorism importance. The MSGPP Protein Production Group (PPG) objectives are: the production of crystallization-optimized proteins for downstream units, the determination of optimal protein production methodologies (Aims 1-3), and the generation of biochemical assays for ligand screening. The PPG has developed and will continue to employ a state-of-the-art, high-throughput protein production protocol, using robotics for cloning, expression and purification. Working closely with the Target Selection and Domain Selection Groups (Project 1), the Protein Characterization and Crystallization Group (Project 3), and the Informatics Group (Core A), a variety of hypotheses will be tested by the PPG to learn which approaches lead to the most successful soluble proteins that give good crystal formation for structure determination. Specific Aim 1 of this project will be to examine the production of soluble proteins in E coli to test the effect of rigorous purification, varying purification tags and refolding techniques for their ability to improve the yield of soluble proteins, crystals, and structures. Specific Aim 2 will be to evaluate the use of variant proteins to improve the number of soluble proteins that produce structures. Specifically, we will test whether varying the length of domain-fragments and site directed mutagenesis leads to increased numbers of soluble proteins, and, ultimately, increased structures. Specific Aim 3 will be to test the hypothesis that expression systems, other than E. coli, can improve the number of soluble proteins produced for successful crystallography. In vitro cell-free protein synthesis, and baculovirus/insect cell culture will be evaluated for their potential improvement in soluble protein, crystals, and structures solved. We estimate the PPG will produce 374 proteins representing 170 different targets from the protozoan pathogens for ligand-based crystallography. The Program Project is estimated to produce 50 new structures of medically-relevant target proteins with 250 structures of these co-crystallized with ligands. These protein-ligand structures will foster structure based drug design for protozoan pathogens of medical and bioterrorism importance.

致病原生动物（MSGPP）的医学结构基因组学的目的是从10个医学和生物恐怖主义重要性的10个原生动物生成药物开发的配体结构信息。 MSGPP蛋白质生产组（PPG）的目标是：用于下游单元结晶优化蛋白的产生，确定最佳蛋白质生产方法（AIMS 1-3）以及生成生化化学测定法进行配体筛选。 PPG已开发并将继续采用最先进的高通量蛋白质生产方案，并使用机器人技术来克隆，表达和纯化。与目标选择和域选择组（项目1），蛋白质表征和结晶组（项目3）和信息学组（核心A）紧密合作，PPG将测试各种假设对于最成功的可溶性蛋白，可以为结构确定提供良好的晶体形成。 该项目的具体目的1将是检查大肠杆菌中可溶性蛋白的产生，以测试严格的纯化，改变纯化标签和重新折叠技术的影响，以提高其提高可溶性蛋白质，晶体和结构的产量。具体目标2将是评估变体蛋白的使用以改善产生结构的可溶性蛋白质的数量。具体而言，我们将测试改变域碎片的长度和定向诱变的长度是否会导致可溶性蛋白质数量增加，并最终导致结构增加。具体目的3将是检验以下假设：除大肠杆菌以外的表达系统可以改善为成功晶体学生产的可溶性蛋白的数量。将评估体外无细胞的蛋白质合成和杆状病毒/昆虫细胞培养 它们在可溶性蛋白质，晶体和结构溶液中的潜在改善。我们估计PPG将产生374个蛋白质，代表原生动物的170个不同靶标 基于配体晶体学的病原体。据估计，该计划项目将产生50个与配体共结晶的250个结构的医学相关目标蛋白的新结构。这些蛋白质 - 配体结构将促进基于结构的药物设计，以实现医学和生物恐怖主义重要性的原生动物病原体。