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

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

Journal of Chromatography A 852(1): 161-173

ISSN/ISBN: 0021-9673

PMID: 10480241

Estrogen receptor-alpha is a member of the nuclear hormone receptor superfamily and is considered as a very important regulatory protein. Human estrogen receptor-alpha has been cloned into Saccharomyces cerevisiae as a fusion to ubiquitin and expression is controlled by a metallothionin promotor. Pilot scale quantities of receptor have been produced by a yeast strain transformed with expression plasmid YEpE13 (Graumann et al., J. Steroid Biochem. Mol. Biol. 57 (1996) 293) in a 14 1 stirred tank reactor. The yeast extract contained 2-4 pmol of receptor protein per mg total protein. A purification scheme has been developed using heparin-affinity chromatography combined with affinity chromatography with immobilized 17beta-estradiol 17-hemisuccinate. Heparin-affinity chromatography was very efficient to remove host cell protein. Accompanying proteins that stabilize unoccupied receptor have not been dissociated during elution. The receptor could be purified 5-10-fold 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 further purified 100-fold with ligand-affinity chromatography using 17beta-estradiol 17-hemisuccinate-bovine serum albumin-Sepharose. The receptor could be kept in its native state, although saturated with 17beta-estradiol. The purification sequence allows an efficient production of receptor. Further improvement of productivity can be only accomplished by increasing the expression level.

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Accession: 010136262

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