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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 
 
 

Expression of human vitamin D receptor in Saccharomyces cerevisiae. Purification, properties, and generation of polyclonal antibodies.

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.[1]

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