Expression of human vitamin D receptor in Saccharomyces cerevisiae. Purification, properties, and generation of polyclonal antibodies.
Year: 1990
Type: article
Abstract: We have cloned a cDNA encoding the human vitamin D receptor (VDR) into a high copy yeast plasmid controlled transcriptionally by the copper-inducible metallothionein (CUP-1) promoter to produce YEpV1. Introduction of this plasmid in the protease deficient Saccharomyces cerevisiae strain BJ 3505 and subsequent growth in the presence of copper and 1,25-dihydroxyvitamin D3 leads to the synthesis of intact VDR comprising over 0.5% of total soluble protein. The VDR was purified to near homogeneity from similarly induced yeast cultures by ammonium sulfate precipitation, and sequential DNA-cellulose and DEAE-Sephadex chromatography, and then characterized for physical and functional properties. The purified VDR associated with a specific synthetic DNA sequence comprising the vitamin D response element as assayed through bandshift analysis. Binding, however, required the presence of a mammalian cell protein factor that also enhances vitamin D response element interaction by mammalian cell-derived VDR. Polyclonal antibodies raised in rabbits against the purified VDR further retarded the receptor/nuclear factor/DNA complex in these analyses. These studies, together with our previous experiments that demonstrate reconstitution of a vitamin D-dependent transcription system in yeast, show that the VDR can be produced and purified from yeast in a functional form.
Source: Journal of Biological Chemistry
Authors: T Sone, D P McDonnell, B W O'Malley, J W Pike
Institution: Baylor College of Medicine
Language: English
Topic: Vitamin D Research Studies
Subfield: Pathology and Forensic Medicine
Field: Medicine
Domain: Health Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W1481038980