Large-scale expression and purification of the human vitamin D receptor and its ligand-binding domain for structural studies

• Published in: Biochemical journal 1999-12, Vol.344 (2), p.297-303
• Main Authors: JUNTUNEN, Kari, ROCHEL, Natacha, MORAS, Dino, VIHKO, Pirkko
• Format: Article
• Language: English
• Subjects: Biochemistry, Molecular Biology; Life Sciences
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3440297

We have expressed recombinant human vitamin D receptor and its ligand-binding domain in Spodoptera frugiperda (Sf9) insect cells with a 30-litre bioreactor. Both proteins were purified to apparent homogeneity with yields of 0.5-3.5 mg/l. Gel-filtration analyses indicated that the purified human vitamin D receptor and its ligand-binding domain were present as monomers in solution. The purified vitamin D receptor and its ligand-binding domain were demonstrated to bind 1α,25-dihydroxyvitamin D3 with high affinity, the Kd values ranging from 0.9 to 1.2 nM. Neutron scattering studies of the ligand-binding domain demonstrated that the samples are homogeneous and contain monomeric species of polypeptides. The purified vitamin D receptor binds to the vitamin D response elements of osteopontin and osteocalcin genes as a homodimer or as a heterodimer with the retinoid X receptor-αδAB and we were able to purify these complexes in quantities sufficient for crystallization studies. The results indicate that we can produce biologically active human vitamin D receptor and its ligand-binding domain in insect cells and purify them for functional and structural studies.