In vitro Evolution of Steroid Receptors to Create Novel Biosensors for Small Organic Molecules

摘要

The overall aim of our research is to demonstrate the feasibility of creating novel biosensors that are targeted against any particular organic compound of interest, and which can be used for rapid and quantitative detection of that compound. The principle of the approach is to use in vitro evolution of an existing polypeptide-based biosensor to select one with a novel specificity. Mutant libraries were created by selectively mutating regions of the receptor. These libraries were screened in a yeast-based system that facilitates the selection of mutant receptors that bind the organic compound of interest. To show proof of principle the libraries were screened for mutant receptors that specifically bind the pesticide, methoxychlor. The unmutated (wild type) receptor does not detectably interact with this compound. Mutants were identified that interact with methoxychlor. Importantly the mutant receptors specifically recognize methoxychlor and not the related compound DDT or the chlorinated hydrocarbon dieldrin. Thus it appears possible to create and identify mutant receptors that specifically recognize the organic compound of interest. The ability of the mutant receptors to activate transcription in response to the target was used to demonstrate the feasibility of a yeast-based biosensor. Recent modifications of the yeast-based screen have dramatically improved the signal-to-noise ratio. Current screening efforts focus on identifying receptors that specifically recognize TNT.