Synthesis and purification of soluble ligand binding domain of the human vitamin D-3 receptor

We expressed and purified milligram quantities of the ligand binding domain of the human 1,25-dihydroxyvitamin D-3 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 degrees 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 D-3 receptor. The 105-427 human 1,25-dihydroxyvitamin D-3 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 D-3 receptor bound 1,25-dihydroxyvitamin D-3 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 D-3 receptor will provide human 1,25-dihydroxyvitamin D-3 receptor ligand binding domain suitable for structural studies. (C) 1996 Academic Press, Inc.