A method for characterization of endogenous ligands to orphan receptors belonging to the steroid hormone receptor superfamily-Isolation of progesterone from pregnancy plasma using progesterone receptor ligand-binding domain

Carol D. Banner, Annika Goos-Nilsson, Jan Sjövall, Jan Åke Gustafsson, Joseph J. Rafter

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

An analytical method is described whereby progesterone is isolated from pregnancy plasma on the basis of the high affinity and specificity of the progesterone receptor for its ligand. Partially purified progesterone receptor ligand-binding domain, expressed as a protein A fusion protein in Escherichia coli, is incubated with a neutral steroid fraction obtained by extraction and ion-exchange chromatography of human late-pregnancy plasma. The incubated sample is passed through two Lipidex 1000 (lipophilic gel) beds. The first, at 4°C, separates the specific ligand-fusion protein complex from nonspecifically bound and unbound compounds, and the second, at 40°C, separates the specific ligand from the protein. Elution of the second bed with methanol yields a fraction containing specific ligand that can be characterized by gas chromatography-mass spectrometry. This methodology may be valuable for identification of endogenous ligands to orphan receptors of the steroid hormone receptor superfamily.

Original languageEnglish (US)
Pages (from-to)163-170
Number of pages8
JournalAnalytical Biochemistry
Volume200
Issue number1
DOIs
StatePublished - Jan 1992

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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