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Expressing the human proteome for affinity proteomics: optimising expression of soluble protein domains and in vivo biotinylation.

Research paper by Tracy T Keates, Christopher D O CD Cooper, Pavel P Savitsky, Charles K CK Allerston, Claire C Phillips, Martin M Hammarström, Neha N Daga, Georgina G Berridge, Pravin P Mahajan, Nicola A NA Burgess-Brown, Susanne S Müller, Susanne S Gräslund, Opher O Gileadi

Indexed on: 27 Oct '11Published on: 27 Oct '11Published in: New Biotechnology



Abstract

The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome.