Conformation-specific affinity purification of proteins using engineered binding proteins: application to the estrogen receptor

Protein Expr Purif. 2006 Jun;47(2):348-54. doi: 10.1016/j.pep.2005.10.021. Epub 2005 Nov 14.


Affinity chromatography coupled with an "affinity tag" has become a powerful and routine technology for the purification of recombinant proteins. However, such tag-based affinity chromatography usually cannot separate different conformational states (e.g., folded and misfolded) of a protein to be purified. Here, we describe a strategy to separate different conformations of a protein by using "tailor-made" affinity chromatography based on engineered binding proteins. Our method involves: (i) engineering of a binding protein specific to a particular conformation of the protein of interest, and (ii) production and immobilization of the binding protein to prepare conformation-specific affinity chromatography media. Using "monobodies," small antibody mimics based on the fibronectin type III domain, as the target-binding proteins, we demonstrated the effectiveness of our method by separating the active form of the estrogen receptor alpha ligand-binding domain (ERalpha-LBD) from a mixture of active and misfolded species and by discriminating two different conformations of ERalpha-LBD bound to different ligands. Our strategy should be generally applicable to the preparation of conformationally homogeneous protein samples.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Chromatography, Affinity
  • Humans
  • Peptide Library*
  • Protein Engineering*
  • Protein Folding*
  • Protein Structure, Tertiary / genetics
  • Receptors, Estrogen / biosynthesis*
  • Receptors, Estrogen / genetics
  • Receptors, Estrogen / isolation & purification
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification


  • Peptide Library
  • Receptors, Estrogen
  • Recombinant Fusion Proteins