Protein Production in the Medical Structural Genomics of Pathogenic Protozoa

致病性原生动物的医学结构基因组学（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。与目标选择和结构域选择组（项目1），蛋白质表征和结晶组（项目3）和信息学组（核心A）密切合作，PPG将测试各种假设，以了解哪些方法导致最成功的可溶性蛋白质，为结构确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的生产，以测试严格纯化、不同纯化标签和重折叠技术对提高可溶性蛋白、晶体和结构产量的能力的影响。具体目标2将是评估使用变异蛋白质来提高产生结构的可溶性蛋白质的数量。具体来说，我们将测试是否不同的结构域片段的长度和定点诱变导致可溶性蛋白质的数量增加，并最终增加结构。具体目标3将是测试表达系统，而不是E。大肠杆菌，可以提高可溶性蛋白质的数量产生成功的晶体学。将评价体外无细胞蛋白质合成和杆状病毒/昆虫细胞培养物， 它们在可溶性蛋白质、晶体和结构方面的潜在改进得到解决。我们估计PPG将产生374种蛋白质，代表原生动物的170种不同靶标 病原体用于配体晶体学。该计划项目预计将产生50种与医学相关的靶蛋白的新结构，其中250种结构与配体共结晶。这些蛋白质-配体结构将促进基于结构的药物设计，用于具有医学和生物恐怖主义重要性的原生动物病原体。

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.