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.
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 degrees C, separates the specific ligand-fusion protein complex from nonspecifically bound and unbound compounds, and the second, at 40 degrees 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.
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Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital, Sweden.
Goos-Nilsson
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A
Sjövall
J
J
Gustafsson
J A
JA
Rafter
J J
JJ
D002852:Chromatography, Ion Exchange; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D008024:Ligands; D011247:Pregnancy / Q000097:blood*; D011374:Progesterone / Q000097:blood*; D011980:Receptors, Progesterone / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D008024:Ligands; D011980:Receptors, Progesterone; D011993:Recombinant Fusion Proteins; D011374:Progesterone; D004247:DNA