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Heterologous Expression of Soluble, Active Proteins inEscherichia coli:The Human Estrogen Receptor Hormone-Binding Domain as Paradigm

Protein Expression and Purification
Publication Date
DOI: 10.1006/prep.2001.1403
  • Heterologous Expression
  • Estrogen Receptor
  • Inclusion Bodies
  • Protein Solubility
  • Ligand-Binding Domain
  • His Tag.
  • Biology
  • Design
  • Physics


Abstract The human estrogen receptor ligand-binding domain (hER-E/F), including the distal F domain, has been expressed to high levels in a soluble, active form in Escherichia coli in order to facilitate biophysical studies. The ability of a series of vectors incorporating strong transcriptional and translational signals to provide an efficient expression system for hER-E/F was investigated. High-level expression was obtained from all of the vectors used in the study. Although the majority of hER-E/F protein was produced in insoluble form under standard bacterial culture conditions, hER-E/F could be produced in soluble, biologically active form by altering the sequence of the expressed protein and by varying the host culture conditions. Several parameters, including the presence of a His tag, growth temperature, and addition of ethanol and 17β-estradiol to the growth medium were shown to have a positive effect on production of soluble hER-E/F. An optimized expression system capable of producing from 25 to 35 mg of biologically active hER-E/F in 1 liter of cell culture was designed, and a simple, rapid purification protocol for hER-E/F produced in this system was developed. Characterization of purified hER-E/F by Edman degradation and mass spectrometry verified the identity of the protein. The K D for 17β-estradiol binding to purified hER-E/F was determined to be 0.6 ± 0.1 nM. The parameters controlling soluble, heterologous protein production observed in this study may be generally applicable to the expression of other heterologous proteins in E. coli.

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