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 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 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 (Kd 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.
Craig
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TA
Nephrology Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Kumar
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D001483:Base Sequence; D002117:Calcitriol / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D011485:Protein Binding; D018167:Receptors, Calcitriol / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins; D012995:Solubility
D017931:DNA Primers; D008024:Ligands; D018167:Receptors, Calcitriol; D011994:Recombinant Proteins; D002117:Calcitriol