Large-scale expression and purification of the human vitamin D receptor and its ligand-binding domain for structural studies.
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 1alpha,25-dihydroxyvitamin D(3) with high affinity, the K(d) 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-alphaDeltaAB 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.
Juntunen
K
K
Biocenter Oulu WHO Collaborating Centre for Research on Reproductive Health, University of Oulu, FIN-90220 Oulu, Finland.
Rochel
N
N
Moras
D
D
Vihko
P
P
D000818:Animals; D001051:Apoenzymes; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002117:Calcitriol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D005822:Genetic Vectors; D020035:Holoenzymes; D006801:Humans; D009502:Neutrons; D015675:Osteocalcin / Q000235:genetics; D053495:Osteopontin; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011485:Protein Binding; D018167:Receptors, Calcitriol / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D020218:Response Elements; D047488:Retinoid X Receptors; D012542:Scattering, Radiation; D012795:Sialoglycoproteins / Q000235:genetics; D018411:Spodoptera / Q000166:cytology; D014157:Transcription Factors / Q000378:metabolism
D001051:Apoenzymes; D004268:DNA-Binding Proteins; D020035:Holoenzymes; D010446:Peptide Fragments; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D047488:Retinoid X Receptors; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D014157:Transcription Factors; D015675:Osteocalcin; D053495:Osteopontin; D002117:Calcitriol