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(136e) Engineering Allosteric Inteins For Use As Biosensors

Gillies, A., Princeton University
Wood, D. W., The Ohio State University

Inteins are intervening protein sequences that post-translationally self-excise from the sequences of various host proteins and are thus protein analogs of self-splicing RNA introns. In most cases, the host protein's activity is controlled by the splicing reaction. It is possible to modify the intein-extein junction residues in such a way that the intein cleaves at one or both of its termini, rather than splices. The ability of inteins to control protein activity has important applications in biosensing and pharmaceutical technologies. This project focuses on engineering allosteric inteins that can be controlled by the presence of small molecule ligands. These inteins are created by inserting small-molecule recognition and binding domains into the intein. In these constructs, the conformational change in the recognition domain upon ligand binding is coupled to a conformational change in the intein that affects its activity. The binding domain-intein complex is coupled to a thymidylate synthase selection system that allows the intein's activity to be monitored in Escherichia coli cells. Hormone-sensing inteins that detect estrogen receptor agonists and antagonists, as well as compounds that target the thyroid hormone receptor, have previously been developed. These sensors have been used successfully to identify estrogenic compounds in synthetic small-molecule libraries as well as plant extracts, and show particular promise in the detection of weakly binding compounds such as environmental pollutants. The intein-based sensors have a modular design, which allows the rapid development of new sensors by swapping the ligand binding domains. For example, an arabinose-sensing splicing intein has been created by inserting the E. coli arabinose binding domain into the intein instead of the hormone binding domains previously reported. The arabinose-sensing intein is characterized by a marked increase in growth of thymidylate synthase knockout E. coli cells in the presence of arabinose at room temperature. This biosensor is being further developed using mutated arabinose binding domains to sense other ligands, such as lactate, TNT, and serotonin.