The expression of milligram amounts of functional human 1,25-dihydroxyvitamin D3 receptor in a bacterial expression system.
We expressed milligram amounts of functional human 1,25-dihydroxyvitamin D3 receptor in a bacterial expression system in which the cloned cDNA for the hVDR was expressed under the control of bacterial T7 polymerase. The hVDR protein comprised approximately 60% of total bacterial protein. It migrated on polyacrylamide-sodium dodecyl sulfate gels with an M(r) of 48,000. It had the predicted amino acid composition and amino acid sequence analysis. The expressed protein was bound by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) with a Kd in the nanomolar range. It sedimented on sucrose density gradients at 3.5S. Furthermore, the expressed protein bound to the osteocalcin vitamin D response element (VDRE) as assessed by a gel mobility shift assay. The expression of large amounts of hVDR protein should allow for the use of this protein in structure-function and x-ray crystallography studies.
Kumar
R
R
Nephrology Research Unit, Mayo Clinic, Rochester, MN 55905.
Schaefer
J
J
Wieben
E
E
D000595:Amino Acid Sequence; D001483:Base Sequence; D002117:Calcitriol / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D009838:Oligodeoxyribonucleotides; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D002117:Calcitriol