Bacterial expression and characterization of the ligand-binding domain of the vitamin D receptor

The ligand-binding domain of the rat vitamin D receptor (amino acids 115-42 3) was expressed as an amino-terminal His-tagged protein in a bacterial exp ression system and purified over Ni-nitrilotriacetic acid resin and a Mono S column. The purified protein bound its Ligand, 1,25-dihydroxyvitamin D-3, with high affinity, similar to that of the full-length protein. Saturation of the protein with Ligand quenched 90% of the tryptophan fluorescence, co nsistent with the purified protein being uniformly able to bind ligand. Add ition of ligand produced no change in the tryptophan fluorescence lifetime, suggesting static quenching as the mechanism of fluorescence decrease. The near-UV circular dichroism spectrum showed a large increase in signal foll owing the addition of ligand, consistent with a change in the environment o f aromatic amino acid side chains. The far-UV circular dichroism spectrum w as consistent with a protein of high alpha-helical content. Sedimentation e quilibrium experiments demonstrated that the protein formed higher-order co mplexes, and the distribution of the protein among these complexes was sign ificantly shifted by addition of ligand. (C) 1999 Academic Press.