W0416

Center for Eukaryotic Structural Genomics: Our Pipeline for Protein Production. Han, Byung Woo¹, Aceti, Dave¹, Bingman, Craig¹, Blommel, Paul¹, Dyer, Dave¹, Fox, Brian¹, Fredrick, Ronnie¹, Hegeman, Adrian¹, Jeon, Won Bae¹, Johnson, Ken¹, Kimball, Todd¹, Liesman, Scott¹, Markley, John¹, Narayama, Ramya¹, Newman, Craig¹, Phillips, George¹, Rayment, Ivan¹, Seder, Kory¹, Smith, David¹, Sreenath, Hassan¹, Sussman, Mike¹, Thao, Sandy¹, Ulrich, Eldon¹, Vinaraov, Dmitriy¹, Wrobel, Russell¹, Zhao, Qin¹, Zolnai, Zsolt¹, Volkman, Brian², Peterson, Francis², Lytle, Betsy², Dunker, Keith³, Linial, Michal⁴, Endo, Yaeta⁵, Kainosho, Masatsune⁶, ¹Univ. of Wisconsin, Madison, ²Medical College of Wisconsin, Milwaukee, ³Molecular Kinetics, Pullman, Washington, ⁴Hebrew Univ., Jerusalem, Israel, ⁵Ehime Univ., Matsuyama, Japan, ⁶Tokyo Metropolitan Univ., Tokyo, Japan.

The Center for Eukaryotic Structural Genomics (CESG) was founded as a collaborative effort to develop critical technologies for determining three-dimensional structures of proteins rapidly and economically. CESG’s initial focus is on the genome of the model plant Arabidopsis thaliana. CESG has developed its own laboratory information management system (‘Sesame’) designed to track and evaluate steps in the process leading from gene to published structure. CESG’s software periodically analyzes the entire Arabidopsis genome to determine targets to be produced. Priority is given to targets likely to open up important regions of conformational space or to elucidate novel fold-function relationships. CESG also considers proteins of structural interested by the plant science community. Gene chips produced by maskless array DNA synthesis are being used to determine the presence of targets in cDNA pools generated by RT-PCR of RNA isolated from an Arabidopsis callus cell line. CESG’s standard pipeline protocol utilizes Invitrogen’s ‘Gateway’ plasmid construction system in 96 well plates, expression and solubility assays, large-scale E. coli fermentation in 11 disposable bottles, TEV protease-cleavable tags, semi-automated purification technology, and robotics-based crystallization. Efforts are also underway to produce protein by cell-free methods for efficient isotopic labeling for NMR structure determination. Preliminary results on the cloning, expression, solubility, and structural characterization of targets on both bacterial and cell-free systems will be presented at the meeting. Additional information can be found at http://uwstructuralgenomics.org/.