Affinity chromatography of human estrogen receptor-alpha expressed in Saccharomyces cerevisiae. Combination of heparin- and 17beta-estradiol-affinity chromatography.
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 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 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.
Feng
W
W
Institute for Applied Microbiology, University of Agricultural Sciences, Vienna, Austria.
Graumann
K
K
Hahn
R
R
Jungbauer
A
A
D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000737:chemistry*; D047628:Estrogen Receptor alpha; D006493:Heparin / Q000737:chemistry*; D006801:Humans; D011960:Receptors, Estrogen / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D017931:DNA Primers; D047628:Estrogen Receptor alpha; D011960:Receptors, Estrogen; D011994:Recombinant Proteins; D004958:Estradiol; D006493:Heparin