Core B, Abstract The overall goal of this program project is to understand key pathways in the biology of Mtb that are important in the pathogen?s adaptation to disease-relevant stress conditions ? including moving from genetic and molecular characterization to a biochemical understanding of function. Ultimately this requires both purified proteins, and enzymatic or binding assays. The Sacchettini lab has considerable experience with both protein expression, and purification as well as with developing biochemical assays to investigate protein function. While they usually focus on essential enzymes, the methods used can be applied to most biological macromolecules. The Sacchettini laboratory also serves as an important interface to the TB Structural Genomics Consortium (TBSGC) and when appropriate they will provide access to structural resources in order to help determine function. They will produce recombinant protein and develop assays for all projects within the program project, utilizing methods developed in the lab or published methods. For those cases where there is no first hand or published information, they will use their general strategy for making soluble and active Mtb proteins, that they have adopted from their structural genomics pipeline for high throughout cloning, expression and purification. The Core?s work on defining the biochemical function of the target proteins will rely heavily on interactions with the metabolomics core (Core A). This will not be an obstacle as these labs currently work closely on several projects. In addition, the Core will use standard bioinformatic methods to help understand what the amino sequence suggests about function, in addition to structures of similar proteins. The core will perform two main activities: Activity 1: Clone, overexpress and purify recombinant Mtb proteins for functional analysis using in vitro biochemical characterization and assays. Activity 2: Develop in vitro biochemical assays for project proteins.
