Protein Production in the Medical Structural Genomics of Pathogenic Protozoa

致病原生动物的医学结构基因组学（MSGPP）旨在从10种具有医学和生物恐怖主义重要性的原生动物中获得药物开发的配体结构信息。MSGPP蛋白质生产组（PPG）的目标是：为下游单元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分析。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，使用机器人技术进行克隆、表达和纯化。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，PPG将测试各种假设，以了解哪种方法可以获得最成功的可溶性蛋白质，这些蛋白质可以为结构确定提供良好的晶体形成。

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.