Directed evolution of estrogen receptor proteins with altered ligand-binding specificities

Kazi Mohammed Didarul Islam, Meik Dilcher, Corinna Thurow, Carsten Vock, Ilga Kristine Krimmelbein, Lutz Friedjan Tietze, Victor Gonzalez, Huimin Zhao, Christiane Gatz

Research output: Contribution to journalArticle


Transcriptional activators that respond to ligands with no cellular targets are powerful tools that can confer regulated expression of a transgene in almost all biological systems. In this study, we altered the ligand-binding specificity of the human estrogen receptor α (hERα) so that it would recognize a non-steroidal synthetic compound with structural similarities to the phytoestrogen resveratrol. For this purpose, we performed iterative rounds of site-specific saturation mutagenesis of a fixed set of ligand-contacting residues and subsequent random mutagenesis of the entire ligand-binding domain. Selection of the receptor mutants and quantification of the interaction were carried out by exploiting a yeast two-hybrid system that reports the ligand-dependent interaction between hERα and steroid receptor coactivator-1 (SRC-1). The screen was performed with a synthetic ligand (CV3320) that promoted growth of the reporter yeast strain to half maximal levels at a concentration of 3.7 μM. The optimized receptor mutant (L384F/L387M/Y537S) showed a 67-fold increased activity to the synthetic ligand CV3320 (half maximal yeast growth at 0.055 μM) and a 10-fold decreased activity to 17ß-estradiol (E2; half maximal yeast growth at 4 nM). The novel receptor-ligand pair partially fulfills the requirements for a specific 'gene switch' as it responds to concentrations of the synthetic ligand which do not activate the wildtype receptor. Due to its residual responsiveness to E2 at concentrations (4 nM) that might occur in vivo, further improvements have to be performed to render the system applicable in organisms with endogenous E2 synthesis.

Original languageEnglish (US)
Pages (from-to)45-52
Number of pages8
JournalProtein Engineering, Design and Selection
Issue number1
StatePublished - Jan 1 2009


  • Estrogen receptor
  • Ligand specificity
  • Saturation mutagenesis

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology
  • Bioengineering
  • Molecular Biology

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  • Cite this

    Islam, K. M. D., Dilcher, M., Thurow, C., Vock, C., Krimmelbein, I. K., Tietze, L. F., Gonzalez, V., Zhao, H., & Gatz, C. (2009). Directed evolution of estrogen receptor proteins with altered ligand-binding specificities. Protein Engineering, Design and Selection, 22(1), 45-52.