title: Gene design, cloning and protein-expression methods for high-value targets at the Seattle Structural Genomics Center for Infectious Disease.
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.
authors: Seattle Structural Genomics Center for Infectious Disease (SSGCID), USA. araymond@embios.com
authors: Amy
authors: A
authors: Raymond
authors: Taryn
authors: T
authors: Haffner
authors: Nathan
authors: N
authors: Ng
authors: Don
authors: D
authors: Lorimer
authors: Bart
authors: B
authors: Staker
authors: Lance
authors: L
authors: Stewart
mesh_terms: D003001:Cloning, Molecular; D018360:Crystallography, X-Ray / Q000379:methods*; D023281:Genomics; D053118:Influenza A Virus, H1N1 Subtype / Q000201:enzymology; D008958:Models, Molecular; D009976:Orthomyxoviridae Infections; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics; D014861:Washington
chemical_list: C057027:PB2 protein, Influenzavirus A; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase