Cloning the human vitamin D receptor into the pTwin-1 expression vector

Abstract

Many of the actions of 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3] are mediated by binding to the nuclear vitamin D receptor (VDR). VDR is a member of a superfamily of nuclear receptors that are ligand-dependent transcription factors. Ligand binding induces conformational changes in the VDR that enable the receptor to interact with other coactivators to modulate gene transcription. In order to better characterize the binding of the VDR to 1,25(OH) 2D 3 and to analogs of 1,25(OH) 2D 3, we have cloned the cDNA for the human VDR into the pTwin1 expression system. The expression system results in the cDNA for a chitin-binding peptide and a yeast intein fused in frame with the N-terminal end of the cDNA for VDR. The intein cDNA codes for a self-cleaving peptide that can release VDR, without any additional amino acids, from a chitin column by changing the pH of the buffer. Western blot analysis of the VDR-fusion protein indicates that a protein of approximately 75 kDA was obtained as expected.