Genomes-to-Life Protein Complex Isolation Capability
EMSL Project ID
6504
Abstract
We have established a pilot proteomics facility in support of the "Goal 1" of the Department of Energy's Genomes to Life program, which seeks to identify 80% of the protein complexes in a cell under a specific set of conditions. The primary aim of this facility is to develop new approaches for rapid, robust isolation of protein complexes as well as develop methods to analyze protein complexes, including a determination of their component stoichiometry, modifications, and relative levels. We are also examining spatial and temporal distributions of protein complexes within microorganisms. An important part of this research program is the integration of computational tools that will combine experimental information with proteomics data. Three primary approaches are being taken to isolate protein complexes: 1) expression of epitope-tagged proteins in target cells to allow direct isolation, 2) exogenous expression of tagged proteins to be used as "bait" in equilibrium exchange experiments, 3) generation of affinity reagents using yeast surface display technology to allow isolation of native complexes. To date, we have cloned and expressed over 20 proteins from Shewanella oneidensis to use in our initial screenings and have built an appropriate bioinformatics infrastructure to track sample handling and manage the large amounts of data generated by this project. We are also exploring high-throughput, automated approaches to sample preparation, cross-linking and mass spectrometry analysis. The work in this project is intended to provide the technological foundation for future proteomics core facilities. Supported by the U.S. Department of Energy.
Project Details
Project type
Exploratory Research
Start Date
2004-01-09
End Date
2007-01-14
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Mayer MUljana, L Shi, and TC Squier. 2005. "One-step, non-denaturing isolation of an RNA polymerase enzyme complex using an improved multi-use affinity probe resin." Molecular Biosystems 1(1):53-56.