Versatile copurification procedure for rapid isolation of homogeneous RAR-RXR heterodimers.
RAR-RXR heterodimeric complexes (RARalphaDeltaAB-RXRalphaDeltaAB) bound to cognate DR5 DNA response elements were purified to apparent structural and functional homogeneity using a novel versatile immobilized metal affinity chromatography (IMAC) copurification procedure. Dynamic light scattering studies indicated that the complexes were more than 85% monodisperse. By electron microscopy the negatively stained RAR-RXR-DNA complexes appeared homogeneous and corresponded to a dimeric arrangement of the molecules. Using heterodimers purified according to this procedure we demonstrate ligand binding of RXR in the context of the RAR-RXR heterodimer-DNA complex. The present copurification procedure is rapid and has yielded high quality heterodimer-DNA complexes suitable for both structural and biochemical studies.
Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC)/CNRS/INSERM/ULP, C.U. de Strasbourg, Illkirch Cedex, 67404, France.
J
J
Iyer
D
D
Bonnier
F
F
Granger
C
C
Iltis
V
V
Andry
P
P
Schultz
H
H
Gronemeyer
D002846:Chromatography, Affinity; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D008024:Ligands; D008854:Microscopy, Electron; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D008024:Ligands; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors