TY - JOUR
T1 - Synthesis and purification of soluble ligand binding domain of the human vitamin D3 receptor
AU - Craig, Theodore A.
AU - Kumar, Rajiv
N1 - Funding Information:
1Address all inquires to Dr. Rajiv Kumar, Mayo Clinic, 200 First St., SW., 911A Guggenheim Bldg., Rochester, MN 55905. Fax: 507-266-4710; e-mail: rkumar@mayo.edu. 2Supported by NIH Grant DK 25409.
PY - 1996/1/26
Y1 - 1996/1/26
N2 - 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.
AB - 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.
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U2 - 10.1006/bbrc.1996.0160
DO - 10.1006/bbrc.1996.0160
M3 - Article
C2 - 8579612
AN - SCOPUS:0030048280
SN - 0006-291X
VL - 218
SP - 902
EP - 907
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 3
ER -