ONE-STEP IMMUNOAFFINITY PURIFICATION OF RECOMBINANT HUMAN RETINOIC ACID RECEPTOR-GAMMA

Retinoic acid receptors (RAR) are members of the steroid/thyroid hormo ne receptor superfamily and serve as ligand-activated transcription fa ctors. In order to facilitate studies of receptor protein, we have gen erated a monoclonal antibody to the human RAR gamma, and have develope d a procedure to purify the full-length receptor expressed in insect c ells. The monoclonal antibody (A10) was developed using as antigen a c arboxy-terminal fragment of the human RAR gamma expressed as a bacteri al fusion protein. The A10 monoclonal antibody binds to both native an d denatured forms of the human RAR gamma. This antibody was immobilize d on a resin and used to purify full-length, baculo-virus-expressed hu man RAR gamma to near homogeneity. The immunoaffinity-purified recepto r is >90-95% pure as revealed by silver-stained gels. The identity of the single protein band as RAR gamma was verified by immunoblotting us ing a polyclonal antibody to an epitope distinct from that recognized by the A10 antibody. The pure human RAR gamma is functional with respe ct to both ligand and DNA binding. Scatchard analysis of H-3-labeled a ll-trans retinoic acid binding to purified human RAR gamma revealed a single, high-affinity binding site with a K-d of similar to 2 nM. Bind ing of the pure RAR gamma to a DR5-type retinoic acid response element was also studied. Response element binding by RAR gamma required the presence of the retinoid X receptor, but did not require the presence of additional proteins. Human RAR gamma protein purified in this fashi on will be useful in future structural and functional studies. (C) 199 7 Academic Press.