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Regulation of protein activity with small-molecule-controlled inteins

Department of Chemical Engineering, Princeton University, Princeton, New Jersey 08544, USA

(RECEIVED July 18, 2004; FINAL REVISION October 22, 2004; ACCEPTED October 27, 2004)

Inteins are the protein analogs of self-splicing RNA introns, as they post-translationally excise themselves from a variety of protein hosts. Intein insertion abolishes, in general, the activity of its host protein, which is subsequently restored upon intein excision. These protein elements therefore have the potential to be used as general molecular "switches" for the control of arbitrary target proteins. Based on rational design, an intein-based protein switch has been constructed whose splicing activity is conditionally triggered in vivo by the presence of thyroid hormone or synthetic analogs. This modified intein was used in Escherichia coli to demonstrate that a number of different proteins can be inactivated by intein insertion and then reactivated by the addition of thyroid hormone via ligand-induced splicing. This conditional activation was also found to occur in a dose-dependent manner. Rational protein engineering was then combined with genetic selection to evolve an additional intein whose activity is controlled by the presence of synthetic estrogen ligands. The ability to regulate protein function post-translationally through the use of ligand-controlled intein splicing will most likely find applications in metabolic engineering, drug discovery and delivery, biosensing, molecular computation, as well as many additional areas of biotechnology.

Keywords: protein function regulation; intein; protein splicing; allosteric enzyme; domain insertion; small molecule

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04996905.

Reprint requests to: David W. Wood, Department of Chemical Engineering, Princeton University, Engineering Quadrangle, Olden St., Princeton, NJ 08544, USA; e-mail: dwood{at}princeton.edu; fax: (609) 258-0211.

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