High activity, soluble, bacterially expressed human vitamin D receptor and its ligand binding domain.
The effects of 1 alpha, 25(OH)2 vitamin D3 on cell growth and differentiation are primarily mediated by the nuclear vitamin D receptor (VDR). In order to study aspects of receptor function and ultimately the structural basis of the VDR-ligand interaction, it is necessary to produce large quantities of purified VDR. To achieve this, we have expressed the human VDR and its ligand binding domain in E. coli as fusion proteins with the maltose binding protein using the expression vector pMal-c2. In this system high level expression of both fusion proteins in a soluble form was achieved, whereas previous attempts to express the VDR in E. coli have resulted in an insoluble product. After affinity purification on amylose resin, the fusion proteins were isolated with yields of 10-20 mg/l of culture. Both forms of the recombinant receptor bound 1 alpha, 25(OH)2 vitamin D3 with high affinity; estimated Kd values from Scatchard analysis for the purified full-length receptor and the ligand binding domain were 0.16 +/- 0.07 nM and 0.04 +/- 0.02 nM, respectively. The nonhypercalcemic analogs of vitamin D, MC903 and delta 22-1, 25S, 26 (OH)3 vitamin D3, bound the recombinant fusion proteins with a similar affinity to the native ligand, 1 alpha, 25(OH)2 vitamin D3. In addition, the full-length VDR fusion protein was shown by gel shift analysis to bind weakly to the human osteocalcin gene vitamin D response element, an interaction greatly facilitated by addition of RXR alpha. These results show that the bacterial expression system detailed here is readily able to produce soluble and functional VDR and its ligand binding domain in high yield. These proteins are easily purified and should be suitable for further structural and functional analysis.
Mottershead
D G
DG
CRC for Biopharmaceutical Research, Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, N.S.W., Australia.
Polly
P
P
Lyons
R J
RJ
Sutherland
R L
RL
Watts
C K
CK
D001667:Binding, Competitive; D002846:Chromatography, Affinity; D005786:Gene Expression Regulation; D006801:Humans; D008024:Ligands; D010957:Plasmids; D011485:Protein Binding; D018167:Receptors, Calcitriol / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011995:Recombination, Genetic
D008024:Ligands; D018167:Receptors, Calcitriol; D011993:Recombinant Fusion Proteins