Purification of human estrogen receptor expresed in sccharomyces cerevisiae by affinity chromatography

摘要

Human estrogen receptor, a 65 kD protein has been cloned into yeast (Saccharomyces cerevisiae) by fusion to ubiquitin. Expression is controlled by a metallothionin promotor. In vivo the protein is associated with various chaperones, such as hsp90, hsp70 and p56. After activation by binding to the cognate hormone or a mimic of the hormone, the receptor sheds the chaperones and traffics into the nucleus, where it binds to its hormone response eement and subsequent transactivation of the response gene. When the receptor is overexpressed in the yeast, the protein is presnet in the cytoplasm associated with respective chaperones. Purification strategies for the human estrogen receptors must consider the protein complexes formed after expression. When the compelx dissociates the protein becomes unstable and tends to agregate. Furthermore the proteolytic activity degraded the receptor. Purification of human estrogen receptor using ion-exchange chromatography, heparin-affinity chromatogaphy, affinity-chromatography with immobilized 17-beta estradiol is described the combinations of the aforementioned methods will be shown and discussed.