One-step immunoaffinity purification of recombinant human retinoic acid receptor gamma.
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 RAR gamma, 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 RAR gamma expressed as a bacterial fusion protein. The A10 monoclonal antibody binds to both native and denatured forms of the human RAR gamma. This antibody was immobilized on a resin and used to purify full-length, baculovirus-expressed human RAR gamma 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 RAR gamma was verified by immunoblotting using a polyclonal antibody to an epitope distinct from that recognized by the A10 antibody. The pure human RAR gamma is functional with respect to both ligand and DNA binding. Scatchard analysis of 3H-labeled all-trans retinoic acid binding to purified human RAR gamma revealed a single, high-affinity binding site with a Kd of approximately 2 nM. Binding 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 fashion will be useful in future structural and functional studies.
Repa
J J
JJ
Interdepartmental Graduate Program in Nutritional Sciences, University of Wisconsin-Madison 53706, USA.
Berg
J A
JA
Kaiser
M E
ME
Hanson
K K
KK
Strugnell
S A
SA
Clagett-Dame
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D051379:Mice; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D051381:Rats; D018168:Receptors, Retinoic Acid / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D018411:Spodoptera; D000097768:Retinoic Acid Receptor gamma
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; D000097768:Retinoic Acid Receptor gamma