title: Production and characterization of a retinoic acid receptor RARγ construction encompassing the DNA binding domain and the disordered N-terminal proline rich domain.
abstract: Gene activation by retinoic acid nuclear receptors (RAR) is regulated by a number of molecular events such as ligand binding, interaction with cognate DNA sequences and co-regulatory proteins, and phosphorylation. Among the several phosphorylation sites that are involved in the non-genomic regulatory pathways of the RAR, two are located in a proline rich domain (PRD) within the N-terminal domain (NTD) of the receptor. This region is predicted to be intrinsically disordered, complicating its production and purification. We present here an approach enabling the high yield production of RAR fragments encompassing the PRD and the DNA binding domain (DBD). We found that expression levels were dependent on where the position of the N-terminal boundary of the fragment was placed within the RAR sequence. The purification protocol involves the use of maltose binding protein as a solubilising tag and extensive centrifugation steps at critical points of the purification process. This protocol is suitable to express (15)N, (13)C labeled proteins enabling nuclear magnetic resonance studies. The resulting proteins were characterized by biophysical methods including Small Angle X-ray Scattering and NMR. These studies showed that PRD extension of RARγ is disordered in solution, a state that is compatible with modifications such as phosphorylation.
authors: Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université de Strasbourg/CNRS UMR 7104/INSERM U964, 1 rue Laurent Fries, 67404 Illkirch, France.
authors: Denise
authors: D
authors: Martinez-Zapien
authors: Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université de Strasbourg/CNRS UMR 7104/INSERM U964, 1 rue Laurent Fries, 67404 Illkirch, France.
authors: Marc-André
authors: MA
authors: Delsuc
authors: Equipe Oncoprotéines, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brandt, BP10413, 67412 Illkirch Cedex, France.
authors: Gilles
authors: G
authors: Travé
authors: Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université de Strasbourg/CNRS UMR 7104/INSERM U964, 1 rue Laurent Fries, 67404 Illkirch, France.
authors: Régis
authors: R
authors: Lutzing
authors: Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université de Strasbourg/CNRS UMR 7104/INSERM U964, 1 rue Laurent Fries, 67404 Illkirch, France.
authors: Cécile
authors: C
authors: Rochette-Egly
authors: Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université de Strasbourg/CNRS UMR 7104/INSERM U964, 1 rue Laurent Fries, 67404 Illkirch, France. Electronic address: kieffer@igbmc.fr.
authors: Bruno
authors: B
authors: Kieffer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D064267:Intrinsically Disordered Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011392:Proline; D017434:Protein Structure, Tertiary; D018168:Receptors, Retinoic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D000097768:Retinoic Acid Receptor gamma
chemical_list: D004247:DNA; D064267:Intrinsically Disordered Proteins; D011392:Proline; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D000097768:Retinoic Acid Receptor gamma
keywords: Intrinsically disordered proteins; Proline rich domain; Retinoic acid receptor