One-Step Immunoaffinity Purification of Recombinant Human Retinoic Acid Receptor γ

• Published in: Protein expression and purification 1997-04, Vol.9 (3), p.319-330
• Main Authors: Repa, Joyce J., Berg, Jessica A., Kaiser, Mary E., Hanson, Kristine K., Strugnell, Stephen A., Clagett-Dame, Margaret
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal; Baculoviridae - genetics; Binding Sites; Chromatography, Affinity; Escherichia coli - genetics; Humans; Kinetics; Mice; Peptide Fragments - genetics; Peptide Fragments - immunology; Rats; Receptors, Retinoic Acid - genetics; Receptors, Retinoic Acid - immunology; Receptors, Retinoic Acid - isolation & purification; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - isolation & purification; Retinoic Acid Receptor gamma; Spodoptera
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.1996.0696

Retinoic acid receptors (RAR) are members of the steroid/thyroid hormone receptor superfamily and serve as ligand-activated transcription factors. In order to facilitate studies of receptor protein, we have generated a monoclonal antibody to the human RARγ, and have developed a procedure to purify the full-length receptor expressed in insect cells. The monoclonal antibody (A10) was developed using as antigen a carboxy-terminal fragment of the human RARγ expressed as a bacterial fusion protein. The A10 monoclonal antibody binds to both native and denatured forms of the human RARγ. This antibody was immobilized on a resin and used to purify full-length, baculovirus-expressed human RARγ to near homogeneity. The immunoaffinity-purified receptor is >90–95% pure as revealed by silver-stained gels. The identity of the single protein band as RARγ was verified by immunoblotting using a polyclonal antibody to an epitope distinct from that recognized by the A10 antibody. The pure human RARγ is functional with respect to both ligand and DNA binding. Scatchard analysis of3H-labeled all-transretinoic acid binding to purified human RARγ revealed a single, high-affinity binding site with aKdof ∼2 nm. Binding of the pure RARγ to a DR5-type retinoic acid response element was also studied. Response element binding by RARγ required the presence of the retinoid X receptor, but did not require the presence of additional proteins. Human RARγ protein purified in this fashion will be useful in future structural and functional studies.