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Protein Expr Purif. 2006 Jun;47(2):348-54. Epub 2005 Nov 14.

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

Author information

1
Department of Biochemistry and Molecular Biology, The University of Chicago, IL 60637, USA.

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 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.

PMID:
16325424
DOI:
10.1016/j.pep.2005.10.021
[Indexed for MEDLINE]

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