Affinity chromatography of human estrogen receptor-alpha expressed in Saccharomyces cerevisiae - Combination of heparin- and 17 beta-estradiol-affinity chromatography

Estrogen receptor-alpha is a member of the nuclear hormone receptor superfa mily and is considered as a very important regulatory protein. Human estrog en receptor-alpha has been cloned into Saccharomyces cerevisiae as a fusion to ubiquitin and expression is controlled by a metallothionin promotor. Pi lot scale quantities of receptor have been produced by a yeast strain trans formed with expression plasmid YEpE13 [Graumann et al., J. Steroid Biochem. Mel. Biol. 57 (1996) 293] in a 14 1 stirred tank reactor. The yeast extrac t contained 2-4 pmol of receptor protein per mg total protein. A purificati on scheme has been developed using heparin-affinity chromatography combined with affinity chromatography with immobilized 17 beta-estradiol 17-hemisuc cinate. Heparin-affinity chromatography was very efficient to remove host c ell protein. Accompanying proteins that stabilize unoccupied receptor have not been dissociated during elution. The receptor could be purified 5-10-fo ld in ligand-free state. In contrast to previous reports, we did not find a difference of the binding affinity of liganded and unliganded receptor for heparin immobilized onto Sepharose. The unoccupied receptor could be furth er purified 100-fold with ligand-affinity chromatography using 17 beta-estr adiol 17-hemisuccinate-bovine serum albumin-Sepharose. The receptor could b e kept in its native state, although saturated with 17 beta-estradiol. The purification sequence allows an efficient production of receptor. Further i mprovement of productivity can be only accomplished by increasing the expre ssion level. (C) 1999 Elsevier Science B.V. All rights reserved.