Purification of a human progesterone receptor

Area of Science:

• Endocrinology
• Molecular Biology
• Biochemistry

Background:

• The progesterone receptor (PR) is a crucial nuclear receptor involved in various physiological processes.
• Understanding the PR's structure and function requires pure, well-characterized receptor preparations.
• Previous methods for PR purification were often inconsistent or yielded low purity.

Purpose of the Study:

• To purify the cytoplasmic progesterone receptor from the human uterus to apparent homogeneity.
• To compare different affinity chromatography methods for receptor purification.
• To characterize the purified receptor's biochemical and biophysical properties.

Main Methods:

• Ammonium sulfate fractionation
• Affinity chromatography (conventional and modified)
• Sucrose density gradient centrifugation
• DEAE-cellulose ion-exchange chromatography
• SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
• Photoaffinity labeling

Main Results:

• A modified affinity chromatography method provided more reproducible results than conventional methods.
• Low-capacity resins yielded the highest purification fold.
• The purified progesterone receptor exhibited a sedimentation coefficient of 3.6 S and a molecular weight of 42,000 Da by SDS-PAGE.
• Hormone binding specificity and behavior on ion-exchange resins were consistent with the native receptor.
• Photoaffinity labeling and alkylation experiments confirmed a single labeled protein of Mr 42,000.

Conclusions:

• The purification protocol successfully yielded a homogeneous human uterine progesterone receptor.
• The characterized properties confirm the receptor's identity and integrity were maintained throughout purification.
• The findings provide a reliable method for obtaining pure progesterone receptor for further studies.