Cloning the human vitamin D receptor into the pTwin-1 expression vector.
Many of the actions of 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] 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)2D3 and to analogs of 1,25(OH)2D3, 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.
Collins
Elaine D
ED
Department of Chemistry, San Jose State University, One Washington Square, San Jose, CA 95192-0101, USA. ecollins@science.sjsu.edu
Espinoza
Aileen
A
Le
Lily Thao-Nhi
LT
Kato
Mallory
M
D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D020869:Gene Expression Profiling; D005822:Genetic Vectors; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D018167:Receptors, Calcitriol / Q000235:genetics*
D018076:DNA, Complementary; D018167:Receptors, Calcitriol