Protein Production and Crystallization at the Joint Center for Structural Genomics

By definition, structural genomics centers must be able to address a large number of diverse protein targets. The methods developed should permit parallel and cost-effective processing while allowing for the diverse nature of proteins. Our approach to this problem is a multi-tiered effort where targets are characterized and categorized by behavior and processed in parallel by appropriate methods. The Joint Center for Structural Genomics (JCSG) has applied this tactic to create a fully integrated and scaleable structure determination pipeline. Highlights of the development of the current pipeline for protein production and crystallization are presented here.

[1]  Slawomir K. Grzechnik,et al.  Shotgun crystallization strategy for structural genomics: an optimized two-tiered crystallization screen against the Thermotoga maritima proteome. , 2003, Acta crystallographica. Section D, Biological crystallography.

[2]  A. Deacon,et al.  A scaleable and integrated crystallization pipeline applied to mining the Thermotoga maritima proteome , 2004, Journal of Structural and Functional Genomics.

[3]  G. Rubin,et al.  Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[4]  S A Lesley,et al.  High-throughput proteomics: protein expression and purification in the postgenomic world. , 2001, Protein expression and purification.

[5]  Virgil L. Woods,et al.  Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Virgil L. Woods,et al.  On the use of DXMS to produce more crystallizable proteins: Structures of the T. maritima proteins TM0160 and TM1171 , 2004, Protein science : a publication of the Protein Society.

[7]  R D Klausner,et al.  The mammalian gene collection. , 1999, Science.

[8]  Adam Godzik,et al.  Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[9]  R C Stevens,et al.  High-throughput protein crystallization. , 2000, Current opinion in structural biology.

[10]  Robert B. Russell,et al.  GlobPlot: exploring protein sequences for globularity and disorder , 2003, Nucleic Acids Res..

[11]  John C. Wootton,et al.  Statistics of Local Complexity in Amino Acid Sequences and Sequence Databases , 1993, Comput. Chem..

[12]  J. Hartley,et al.  DNA cloning using in vitro site-specific recombination. , 2000, Genome research.

[13]  Mark Gerstein,et al.  Mining the structural genomics pipeline: identification of protein properties that affect high-throughput experimental analysis. , 2004, Journal of molecular biology.

[14]  Stephen J. Elledge,et al.  The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes , 1998, Current Biology.

[15]  J. Lee,et al.  High-level expression of M13 gene II protein from an inducible polycistronic messenger RNA. , 1985, Gene.

[16]  Thomas Earnest,et al.  Automation of X-ray crystallography , 2000, Nature Structural Biology.