Center for Molecular and Cellular Systems: High-Throughput Identification and Characterization of Protein Complexes
نویسندگان
چکیده
The Center for Molecular and Cellular Systems (CMCS) focuses on Goal 1 of the Genomics:GTL program, which aims to identify and characterize the complete set of protein complexes within a cell to provide a mechanistic basis of biochemical functions. Over the past two years, the CMCS has emphasized developing technologies that can be incorporated into a high throughput “pipeline” for the robust analysis of protein complexes. Several approaches for the isolation and identification of protein complexes from microbial cells were evaluated. Our experience has demonstrated that no single approach will be sufficient to handle the diverse types of complexes present in a cell. Thus, an integrated pipeline has been developed that uses two affinity-based approaches to isolate protein complexes in which tagged proteins are either expressed endogenously or exogenously. The individual technologies have been refined, validated and assembled into a semi-automated pipeline has been in operation for over 30 continuous weeks. A comprehensive laboratory information management system has been developed for sample tracking, process management, and data control. Experiments using over 200 tagged proteins have been conducted using Rhodopseudomonas palustris and Shewanella oneidensis cultures grown under different states. Data from the two types of pull-down approaches have been compared and have been found to provide complementary information. This suggests that both approaches are needed for comprehensive identification of protein complexes.
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