Synthesis and purification of soluble ligand binding domain of the human vitamin D₃ receptor

We expressed and purified milligram quantities of the ligand binding domain of the human 1,25-dihydroxyvitamin D₃ receptor using a glutathione-S-transferase (GST) fusion protein expression system, Amino acids 105-427 were expressed in E. coli as a GST fusion protein at a reduced (20°C) temperature and purified on glutathione sepharose. The fusion protein adsorbed to glutathione sepharose was cleaved with thrombin to yield soluble 105-427 human 1,25-dihydroxyvitamin D₃ receptor. The 105-427 human 1,25-dihydroxyvitamin D₃ receptor was further purified by Mono Q ion exchange chromatography and was characterized as a single band on SDS-polyacrylamide gel electrophoresis. The 105-427 human 1,25-dihydroxyvitamin D₃ receptor bound 1,25-dihydroxyvitamin D₃ with high affinity (K(d) approximately 10^-9 M) and with a binding capacity of 47 pmoles/nmole protein. Large scale expression of 105-427 human 1,25-dihydroxyvitamin D₃ receptor will provide human 1,25-dihydroxyvitamin D₃ receptor ligand binding domain suitable for structural studies.