Conformation-specific affinity purification of proteins using engineered binding proteins: application to the estrogen receptor.
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.
Huang
Jin
J
Department of Biochemistry and Molecular Biology, The University of Chicago, IL 60637, USA.
Koide
Akiko
A
Nettle
Kendall W
KW
Greene
Geoffrey L
GL
Koide
Shohei
S
D002846:Chromatography, Affinity; D006801:Humans; D019151:Peptide Library; D015202:Protein Engineering; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D011960:Receptors, Estrogen / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D019151:Peptide Library; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins