Skip to main content

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

Publication ,  Journal Article
Sone, T; McDonnell, DP; O'Malley, BW; Pike, JW
Published in: J Biol Chem
December 15, 1990

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.

Duke Scholars

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

December 15, 1990

Volume

265

Issue

35

Start / End Page

21997 / 22003

Location

United States

Related Subject Headings

  • Saccharomyces cerevisiae
  • Regulatory Sequences, Nucleic Acid
  • Recombinant Proteins
  • Receptors, Steroid
  • Receptors, Calcitriol
  • Osteocalcin
  • Molecular Weight
  • Humans
  • Gene Expression
  • Electrophoresis, Gel, Two-Dimensional
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Sone, T., McDonnell, D. P., O’Malley, B. W., & Pike, J. W. (1990). Expression of human vitamin D receptor in Saccharomyces cerevisiae. Purification, properties, and generation of polyclonal antibodies. J Biol Chem, 265(35), 21997–22003.
Sone, T., D. P. McDonnell, B. W. O’Malley, and J. W. Pike. “Expression of human vitamin D receptor in Saccharomyces cerevisiae. Purification, properties, and generation of polyclonal antibodies.J Biol Chem 265, no. 35 (December 15, 1990): 21997–3.
Sone T, McDonnell DP, O’Malley BW, Pike JW. Expression of human vitamin D receptor in Saccharomyces cerevisiae. Purification, properties, and generation of polyclonal antibodies. J Biol Chem. 1990 Dec 15;265(35):21997–2003.
Sone T, McDonnell DP, O’Malley BW, Pike JW. Expression of human vitamin D receptor in Saccharomyces cerevisiae. Purification, properties, and generation of polyclonal antibodies. J Biol Chem. 1990 Dec 15;265(35):21997–22003.

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

December 15, 1990

Volume

265

Issue

35

Start / End Page

21997 / 22003

Location

United States

Related Subject Headings

  • Saccharomyces cerevisiae
  • Regulatory Sequences, Nucleic Acid
  • Recombinant Proteins
  • Receptors, Steroid
  • Receptors, Calcitriol
  • Osteocalcin
  • Molecular Weight
  • Humans
  • Gene Expression
  • Electrophoresis, Gel, Two-Dimensional