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

Theodore A. Craig, Rajiv Kumar

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)902-907
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume218
Issue number3
DOIs
StatePublished - Jan 26 1996

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ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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