Pure and functionally homogeneous recombinant retinoid X receptor.
Mouse retinoid X receptor alpha (RXR alpha) lacking the amino-terminal region A/B (RXR alpha delta AB) has been purified to more than 98% purity and functional homogeneity from bacterial and baculovirus-based recombinant expression systems with yields of 2-8 mg/liter of culture. The purified protein is soluble, and fluorescence quenching analysis demonstrated that it binds its cognate ligand 9-cis-retinoic acid (9-cis-RA) stoichiometrically, and with high affinity. Compared with RXR delta AB expressed in COS-1 cells, bacterially and baculovirus-expressed proteins bind approximately 10 and 5 times less efficiently to direct repeat 1 (DR1) DNA elements, respectively, suggesting that animal cell-specific modification of RXR or interaction with other animal cell-specific factors may modulate DNA binding. 9-cis-RA did not stimulate DR1 binding of functional RXR delta AB expressed in Escherichia coli, Sf9 or COS-1 cells. The previously reported ligand effect that can be observed with in vitro made receptor may therefore be a consequence of a conformational stabilization of improperly folded in vitro synthesized protein.
Chen
Z P
ZP
Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS et Unité 184 de Biologie Moléculaire et de Génie Génétique de de l'INSERM, Institut de Chimie Biologique, Faculté de Médecine, Strasbourg, France.
Shemshedini
L
L
Durand
B
B
Noy
N
N
Chambon
P
P
Gronemeyer
H
H
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection; D014212:Tretinoin / Q000378:metabolism
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014212:Tretinoin