Premium
Developing a compound‐specific receptor for bisphenol a by directed evolution of human estrogen receptor α
Author(s) -
Rajasärkkä Johanna,
Hakkila Kaisa,
Virta Marko
Publication year - 2011
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.23214
Subject(s) - mutant , estrogen receptor , receptor , saccharomyces cerevisiae , yeast , biology , flow cytometry , ligand (biochemistry) , mutation , directed evolution , nuclear receptor , bisphenol a , microbiology and biotechnology , chemistry , biochemistry , genetics , gene , transcription factor , organic chemistry , cancer , breast cancer , epoxy
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.