High level expression of biologically active estrogen receptor in Saccharomyces cerevisiae

D. P. McDonnell, Z. Nawaz, C. Densmore, N. L. Weigel, T. A. Pham, J. H. Clark, B. W. O'Malley

Research output: Contribution to journalArticle

107 Scopus citations

Abstract

Biochemical over-expression of the human estrogen receptor was achieved using a Saccharomyces cerevisiae expression system. The receptor was produced as a novel ubiquitin fusion protein. This fusion protein is short lived in the cell and is processed to produced infused receptor shortly after folding. Conventional high copy expression plasmids produced receptor to about 0.04% of the total soluble protein. By incorporating a defective leu2 allele into these vectors, an additional 5-fold increase in receptor production was obtained. The recombinant receptor was undegraded, soluble and biologically active. Conventional methods of disrupting cells using glass beads had a detrimental effect on the ability of the receptor to bind hormone. Enzymatic digestion of the cell wall followed by hypotonic shock liberates the receptor that quantitatively binds estrogen.

Original languageEnglish (US)
Pages (from-to)291-297
Number of pages7
JournalJournal of Steroid Biochemistry and Molecular Biology
Volume39
Issue number3
DOIs
StatePublished - Sep 1991
Externally publishedYes

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ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Endocrinology
  • Clinical Biochemistry
  • Cell Biology

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