Abstract
Any structural genomics endeavor, particularly ambitious ones such as the NIAID-funded Seattle Structural Genomics Center for Infectious Disease (SSGCID) and Center for Structural Genomics of Infectious Disease (CSGID), face technical challenges at all points of the production pipeline. One salvage strategy employed by SSGCID is combined gene engineering and structure-guided construct design to overcome challenges at the levels of protein expression and protein crystallization. Multiple constructs of each target are cloned in parallel using Polymerase Incomplete Primer Extension cloning and small-scale expressions of these are rapidly analyzed by capillary electrophoresis. Using the methods reported here, which have proven particularly useful for high-value targets, otherwise intractable targets can be resolved.
Highlights
The Seattle Structural Genomics Center for Infectious Disease (SSGCID) was established as a collaboration between Seattle BioMed, Emerald BioStructures and the University of Washington in 2007
The primary mission of SSGCID is to establish a resource for gene-to-structure research focused on the structure determination of $400 protein targets from NIAID Category A–C pathogens, as well as organisms causing emerging and re-emerging infectious diseases (Myler et al, 2009)
The design session aligned the primary structure of the target protein with homologous proteins from the Protein Data Bank (PDB), including all secondary-structure and contact information derived from the PDB files
Summary
The Seattle Structural Genomics Center for Infectious Disease (SSGCID) was established as a collaboration between Seattle BioMed, Emerald BioStructures and the University of Washington in 2007. Synthetic genes are cloned via Polymerase Incomplete Primer Extension (PIPE) cloning (Klock et al, 2008) into a T7-based protein-expression vector engineered to donate an amino-terminal hexahistidine-Smt fusion and are expressed in bacterial cells. Tier 3 can be utilized as a salvage strategy for any targets that have failed to produce sufficient soluble protein in Tiers 1 and 2. Tier 3 serves as an efficient entry point to the SSGCID pipeline for eukaryotic/viral community request targets or any target for which the requestor has failed to produce soluble protein in the bacterial platform. F67, 992–997 laboratory communications high-value SSGCID targets and summarize the utility of these vendor and this one synthetic gene served as a template for cloning methods in our consortium.
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