Protein production for structural genomics - Strategies for the next phase

Abstract

Structural genomics (SG) programs aim at establishing efficient pipelines that allow protein structure determination to be attempted on a genome wide scale, at a high pace and at low cost. Structures produced by these programs not only provide fundamental information on molecular mechanisms of well-studied proteins, but can also hint at the functions of previously nonannotated proteins, and can potentially aid in the rational design of drugs. Targets for each of the SG projects have mainly been contained within a genome, focusing on proteins with no or low homology to known structures, but with some emphasis on potential targets for therapeutic interventions. Most current SG projects began by working on prokaryotic proteins, which are significantly easier than eukaryotic proteins to produce in suitable quantity and quality for structural studies. The most urgent technology developments are therefore required for eukaryotic proteins but, of course, such improvements of protein production methodology are likely to significantly improve the success rates of SG projects targeting prokaryotic proteins as well. The cost of producing proteins in quantities needed for structural studies in most current protein production pipelines is relatively high, partially due to extensive manual interventions required for the scale-up process. Therefore, the development and integration of instrumentation in these steps will be essential for cost reduction and efficiency of future large-scale protein production pipelines. Efficient strategies for improving and assessing the quality of proteins produced by SG pipelines will also have great cost benefits by directing only 'high quality' proteins into subsequent SG pipeline steps of crystallisation, crystal optimisation and NMR data collection and interpretation. Intensive efforts are therefore being made to improve strategies for protein production within SG projects (Braun and LaBaer, 2003). Similarly, in the pharmaceutical sector, strategies for the production of specific proteins with improved success rates are urgently needed. Parallel technologies, related to technologies being established in SG programs, are being developed for the pharmaceutical sector. In the present review, progress towards establishing efficient pipelines for producing proteins for structural genomics is assessed with a particular emphasis on emerging technologies in E.coli, including some from our own laboratory that will be particularly useful for producing eukaryotic proteins for SG projects. Important, but less imminent are technologies for eukaryotic expression (Loomis et al. 2005; Geisse and Henke 2005; McCall et al. 2005; Holz et al. 2003) cell free expression (Sawasaki et al. 2002; Shimizu et al. 2001) and refolding of proteins (Trésaugues et al. 2004; Maxwell et al. 2003; Vincentelli et al. 2004), which are not treated in detail in this review. © 2007 Springer.