Baculovirus-mediated expression of the human vitamin D receptor: Functional characterization, vitamin D response element interactions, and evidence for a receptor auxiliary factor

A baculovirus expression vector system (BEVS) was used to overproduce the full-length human vitamin D receptor (hVDR) in Spodoptera frugiperda ovarian cells. hVDR was expressed to a level of 0.5% of the total soluble protein in this system. Western analysis demonstrated that the baculovirus-generated protein had electrophoretic and immunologie properties equivalent to those of hVDR expressed in mammalian cells. The BEVS-derived receptor displayed specificity and high affinity (apparent K_d = 0.7 nM) for the 1,25(OH)₂D₃ ligand. Recombinant hVDR generated a specific protein-DNA complex with a duplex oligomer containing a vitamin D-responsive element (VDRE) in gel mobility shift assays. The intensity of the VDR-VDRE complex was not affected by 1,25(OH)₂D₃. However, the complex exhibited increased mobility in the presence of hormone, possibly the result of a 1,25(OH)₂D₃-dependent conformational change. A nuclear extract obtained from CV-1 cells markedly enhanced the intensity of this VDR·VDRE complex and produced an additional distinct VDR-dependent complex, thus implicating a role for nuclear auxiliary factors in multiple high affinity VDR·VDRE interactions. Finally, methylation interference studies defined the guanine residues contacted when the putative VDR-auxiliary factor complex associates with the rat osteocalcin VDRE; specifically, all of the GC base pairs in the sequence GGGTGAATGAGGACA. Therefore, these results show that the BEV system elicits high level expression of hVDR with critical functional characteristics being preserved.