Proteonomics: High-Throughput Structural Biology—Methods for Cloning, Protein Expression, and Purification

The problems associated with expressing and purifying human proteins, especially in Escherichia coli, the primary host organism for high-throughput (HTP) applications, are welldocumented and have plagued researchers for decades. Low yields due to toxicity, recombinant protein insolubility, and poor purification are just some of the problems that result1 in typical success rates from 2%–20% when expressing eukaryotic proteins in E. coli (Service). HTP structural genomic (SG) projects, such as NIH’s protein structure initiative (PSI) begun in 2000. Initially, the PSI focused on technology development to provide highly automated procedures for cloning, expression testing, protein purification, and protein crystallization, thus addressing production problems by increasing throughput. The development of these techniques has allowed PSI centers and other similar initiatives around the world to deposit over 2,000 novel protein structures in the Protein Dada Bank (PBD) as of January 2006 (PSI, http://www.nigms.nih.gov/Initiatives/PSI/). Nevertheless, despite the expenditure of significant resources,2 the rate of discovery is much less than hoped for at the beginning of the initiative due to bottlenecks at every stage of the pipeline1 as the problems mentioned above persist.