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

Conformation-specific affinity purification of proteins using engineered binding proteins: Application to the estrogen receptor
June 2006
Jin Huanga, Akiko Koidea, Kendall W. Nettleb, Geoffrey L. Greenea, and Shohei Koide
Protein Expression and Purification
Abstract
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 a ligand-binding domain (ERa-LBD) from a mixture of active and misfolded species and by discriminating two different conformations of ERa-LBD bound to different ligands. Our strategy should be generally applicable to the preparation of conformationally homogeneous protein samples.
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