Technology Development Highlights Generated from the Center for Eukaryotic Structural Genomics

Abstract

The Center for Eukaryotic Structural Genomics (CESG) aims to be the leading center for developing and disseminating tested technologies to efficiently solve structures of eukaryotic proteins. We create, evaluate, and optimize innovative protocols for producing eukaryotic proteins in active forms. We seek to improve the efficiency of all stages from target selection-design to three-dimensional structure determination by X-ray crystallography or NMR spectroscopy, including development of bioinformatic techniques and LIMS tools. Using our protein production platform, we refine methods for improving the yield of structures from high-value targets, in particular proteins from humans and other vertebrates. CESG has a substantial outreach component; more than 400 targets from outside requestors have been accepted for study with a structure success rate of 5%, which compares favorably with the eukaryotic success rates for the total PSI effort. These eukaryotic targets frequently present unique challenges. All CESG protein production protocols and Technology Dissemination Reports are accessible through the PSI Knowledgebase: http://kb-psi-structuralgenomics.org/KB/ and CESG's website. Selected technology developments are presented here. These include advances in expression vector design, enhanced methodology for cell based and wheat germ cell-free expression systems, new software to improve the quality and reduce time for structure determination by X-ray crystallography and NMR, and optimized techniques for the production of TEV protease for use in our protein production platform. We actively share our advances with the biotechnology, pharmaceutical, and academic communities through collaborations, oral presentations, peer-reviewed articles, submissions to public databases and material distribution channels, including PepcDB, PDB, BMRB, PSI Materials Repository, and technology transfer workshops. Supported by NIH/NIGMS Protein Structure Initiative grants GM074901 and GM064598. We acknowledge ALL members of the CESG team for their dedicated work.