Author contributions: J. R., K. H., and M. V. designed research; J. R. performed research (J. R. and K. H. performed flow cytometric experiments); J. R. and K. H. analyzed data; J. R. wrote the paper.
Article first published online: 15 JUN 2011
Copyright © 2011 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 108, Issue 11, pages 2526–2534, November 2011
How to Cite
Rajasärkkä, J., Hakkila, K. and Virta, M. (2011), Developing a compound-specific receptor for bisphenol a by directed evolution of human estrogen receptor α. Biotechnol. Bioeng., 108: 2526–2534. doi: 10.1002/bit.23214
The authors declare no conflict of interest.
- Issue published online: 20 SEP 2011
- Article first published online: 15 JUN 2011
- Accepted manuscript online: 26 MAY 2011 09:30AM EST
- Manuscript Accepted: 12 MAY 2011
- Manuscript Revised: 10 MAY 2011
- Manuscript Received: 17 FEB 2011
- Academy of Finland
- Finnish Graduate School in Environmental Science and Technology
- bisphenol A;
- cell-based assay;
- directed evolution;
- estrogen receptor α;
- mutant library screening method
- Protein engineering;
- Environmental biotechnology;
Directed evolution has become a successful approach to alter ligand binding properties of nuclear receptors. In this study, directed evolution was used to generate a mutant human estrogen receptor α library, which was then used to screen for receptors having enhanced responses to the known endocrine-disrupting chemical, bisphenol A (BPA). A single round of multi-site mutation was combined with an efficient positive/negative library screening method in which positive growth-based selection for the desired activity with BPA was combined with flow cytometric removal of cells having undesired activity with the natural ligand, 17β-estradiol. The screening steps were performed in a Saccharomyces cerevisiae yeast strain containing a genome-integrated his3-yEGFP reporter gene fusion construct. A single round of mutation and screening yielded nine mutants with enhanced responses towards BPA but no detectable induction by 17β-estradiol (up to 90 nM). These BPA-specific mutant receptors may prove useful in the field of environmental analytics, where they could be used to monitor and evaluate the proportion of BPA in hormonally active samples. Biotechnol. Bioeng. 2011;108: 2526–2534. © 2011 Wiley Periodicals, Inc.