Developing a Compound-Specific Receptor for Bisphenol A by Directed Evolution of Human Estrogen Receptor alpha

作者：Rajasarkka Johanna^*; Hakkila Kaisa; Virta Marko

来源：Biotechnology and Bioengineering, 2011, 108(11): 2526-2534.

DOI：10.1002/bit.23214

• bisphenol A
• cell-based assay
• directed evolution
• estrogen receptor alpha
• mutant library screening method

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 a 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, 17 beta-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 17 beta-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.

• 出版日期2011-11