Purification of the human RARgamma ligand-binding domain and crystallization of its complex with all-trans retinoic acid.
Fiche publication
Date publication
janvier 1997
Journal
Biochemical and biophysical research communications
Auteurs
Membres identifiés du Cancéropôle Est :
Pr CHAMBON Pierre, Dr GRONEMEYER Hinrich, Dr MORAS Dino, Dr ROCHEL-GUIBERTEAU Natacha, Dr RUFF Marc, Mr LEROUGE Thierry
Tous les auteurs :
Rochel N, Renaud JP, Ruff M, Vivat V, Granger F, Bonnier D, Lerouge T, Chambon P, Gronemeyer H, Moras D
Lien Pubmed
Résumé
A 28-kDa fragment (residues 178-423) of the human retinoic acid receptor gamma, hRARgamma D3E, encompassing the ligand-binding domain (LBD) was overproduced in Escherichia coli and purified as a monomer to more than 95% purity and homogeneity. The Kd for all-trans retinoic acid binding was 0.6 +/- 0.1 nM. Crystals of the LBD complexed with all-trans retinoic acid were grown at pH 7 from sodium acetate in the presence of detergents using the vapor diffusion method. They diffract to 2.0 A using a synchrotron radiation (lambda=0.91 A) and belong to the tetragonal space group P4(1)2(1)2 with unit cell parameters a=b=60.6 A and c=155.3 A, one monomer per asymmetric unit, a solvent content of ca. 33%, and a Vm value of approximately 2 A3/dalton.
Mots clés
Binding Sites, Cloning, Molecular, Crystallization, Crystallography, X-Ray, DNA Primers, Electrophoresis, Polyacrylamide Gel, Escherichia coli, Humans, Kinetics, Peptide Fragments, chemistry, Polymerase Chain Reaction, Receptors, Retinoic Acid, chemistry, Recombinant Proteins, chemistry, Sequence Tagged Sites, Tretinoin, chemistry
Référence
Biochem. Biophys. Res. Commun.. 1997 Jan;230(2):293-6