Affinity chromatography of human estrogen receptor-α expressed in Saccharomyces cerevisiae: Combination of heparin- and 17β-estradiol-affinity chromatography

• Published in: Journal of Chromatography A 1999-08, Vol.852 (1), p.161-173
• Main Authors: Feng, Wenke, Graumann, Klaus, Hahn, Rainer, Jungbauer, Alois
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Cell receptors; Cell structures and functions; Fundamental and applied biological sciences. Psychology; Hormone receptors. Growth factor receptors. Cytokine receptors. Prostaglandin receptors; Molecular and cellular biology; Proteins; Steroid hormone receptors
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(99)00604-4

Estrogen receptor-α is a member of the nuclear hormone receptor superfamily and is considered as a very important regulatory protein. Human estrogen receptor-α 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 l 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 17β-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 17β-estradiol 17-hemisuccinate–bovine serum albumin–Sepharose. The receptor could be kept in its native state, although saturated with 17β-estradiol. The purification sequence allows an efficient production of receptor. Further improvement of productivity can be only accomplished by increasing the expression level.