Structural genomics is emerging as a principal approach to define protein structure-function relationships. To apply this approach on a genomic scale, novel methods and technologies must be developed to determine large numbers of structures. We describe the design and implementation of a high-throughput structural genomics pipeline and its application to the proteome of the thermophilic bacterium Thermotoga maritima. By using this pipeline, we successfully cloned and attempted expression of 1,376 of the predicted 1,877 genes (73%) and have identified crystallization conditions for 432 proteins, comprising 23% of the T. maritima proteome. Representative structures from TM0423 glycerol dehydrogenase and TM0449 thymidylate synthase-complementing protein are presented as examples of final outputs from the pipeline.

Successful protein expression, purification, and crystallization for challenging targets typically requires evaluation of a multitude of expression constructs. Often many iterations of truncations and point mutations are required to identify a suitable derivative for recombinant expression. Making and characterizing these variants is a significant barrier to success. We have developed a rapid and efficient cloning process and combined it with a protein microscreening approach to characterize protein suitability for structural studies. The Polymerase Incomplete Primer Extension (PIPE) cloning method was used to rapidly clone 448 protein targets and then to generate 2143 truncations from 96 targets with minimal effort. Proteins were expressed, purified, and characterized via a microscreening protocol, which incorporates protein quantification, liquid chromatography mass spectrometry and analytical size exclusion chromatography (AnSEC) to evaluate suitability of the protein products for X‐ray crystallography. The results suggest that selecting expression constructs for crystal trials based primarily on expression solubility is insufficient. Instead, AnSEC scoring as a measure of protein polydispersity was found to be predictive of ultimate structure determination success and essential for identifying appropriate boundaries for truncation series. Overall structure determination success was increased by at least 38% by applying this combined PIPE cloning and microscreening approach to recalcitrant targets. Proteins 2008. © 2007 Wiley‐Liss, Inc.