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

Abstract

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.