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 fortheir potential improvement in soluble protein, crystals, and structures solved. We estimate the PPG will produce 374 proteins representing 170 different targets from the protozoanpathogens 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.
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