Expressing the Human Proteome for Affinity Proteomics: Optimising Expression of Soluble Protein Domains and in Vivo Biotinylation

N Biotechnol. 2012 Jun 15;29(5):515-25. doi: 10.1016/j.nbt.2011.10.007. Epub 2011 Oct 19.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biotin / metabolism
  • Biotinylation
  • Chromatography, Affinity / methods*
  • Crystallization
  • Culture Media
  • Genes
  • Humans
  • Mass Spectrometry
  • Plasmids / metabolism
  • Protein Structure, Tertiary
  • Proteome / chemistry*
  • Proteome / genetics
  • Proteome / isolation & purification
  • Proteome / metabolism*
  • Proteomics / methods*
  • Solubility

Substances

  • Culture Media
  • Proteome
  • Biotin