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.
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Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030.
McDonnell
D P
DP
O'Malley
B W
BW
Pike
J W
JW
D002117:Calcitriol / Q000502:physiology*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D015180:Electrophoresis, Gel, Two-Dimensional; D015870:Gene Expression; D006801:Humans; D008970:Molecular Weight; D015675:Osteocalcin / Q000235:genetics; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology; D011994:Recombinant Proteins / Q000302:isolation & purification; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics
D004268:DNA-Binding Proteins; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D015675:Osteocalcin; D004247:DNA; D002117:Calcitriol