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 "tailormade" 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 (ER alpha-LBD) from a mixture of active and misfolded species and by discriminating two different conformations of ER alpha-LBD bound to different ligands. Our strategy should be generally applicable to the preparation-of conformationally homogeneous protein samples. (c) 2005 Elsevier Inc. All rights reserved.