Purification of a human progesterone receptor

Abstract

The cytoplasmic progesterone receptor from human uterus was purified to apparent homogeneity by a combination of ammonium sulfate fractionation and affinity chromatography. Affinity resins prepared by conventional means were compared to those prepared by a modified method. The latter gave more reproducible results. A consistent finding was that low capacity resins gave the highest fold purification of the receptor. The pure receptor sedimented at 3.6 S on sucrose density gradient centrifugation, was eluted as a single band by 0.2 M KCl from DEAE-cellulose and migrated as a single band of MW 42,000 on NaDodSO4-polyacrylamide gel electrophoresis. MW determinations, obtained from Stokes'' radii and sucrose gradient centrifugation, the receptors'' behavior on ion exchange resins and hormone binding specificity were all similar to those of the receptor found in crude cytosol. When the crude cytosol receptor was photoaffinity labeled by using 3H-labeled 17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione followed by NaDodSO4-polyacrylamide gel electrophoresis, only protein of MW 42,000 was labeled. Alkylation of the pure receptor using 11-deoxycorticosterone bromo[3H]acetate showed labeling of a single protein of MW 42,000. These properties confirm that the identity and integrity of the receptor were maintained throughout its purification.