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.

致病性原生动物医学结构基因组学 (MSGPP) 的目标是从 10 种具有医学和生物恐怖主义重要性的原生动物中产生用于药物开发的配体结构信息。 MSGPP 蛋白质生产小组 (PPG) 的目标是：为下游单位生产结晶优化的蛋白质、确定最佳蛋白质生产方法（目标 1-3）以及生成用于配体筛选的生化测定。 PPG 已经开发并将继续采用最先进的高通量蛋白质生产方案，使用机器人进行克隆、表达和纯化。 PPG 将与目标选择和结构域选择小组（项目 1）、蛋白质表征和结晶小组（项目 3）以及信息学小组（核心 A）密切合作，对各种假设进行测试，以了解哪种方法能够最成功地产生可溶性蛋白质，从而为结构测定提供良好的晶体形成。 该项目的具体目标 1 将是检查大肠杆菌中可溶性蛋白质的产生，以测试严格纯化、不同纯化标签和重折叠技术提高可溶性蛋白质、晶体和结构产量的能力的效果。具体目标 2 是评估变体蛋白质的用途，以提高产生结构的可溶性蛋白质的数量。具体来说，我们将测试改变结构域片段的长度和定点诱变是否会导致可溶性蛋白质数量增加，并最终增加结构。具体目标 3 将是检验以下假设：除大肠杆菌之外的表达系统可以提高成功晶体学产生的可溶性蛋白质的数量。将评估体外无细胞蛋白质合成和杆状病毒/昆虫细胞培养 他们在可溶性蛋白质、晶体和结构方面的潜在改进得到了解决。我们估计 PPG 将产生 374 种蛋白质，代表原生动物的 170 种不同目标 用于基于配体的晶体学的病原体。该计划项目预计将产生 50 种新结构的医学相关目标蛋白，其中 250 种结构与配体共结晶。这些蛋白质配体结构将促进针对具有医疗和生物恐怖主义重要性的原生动物病原体的基于结构的药物设计。