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J Am Chem Soc. 2009 Oct 28;131(42):15284-90. doi: 10.1021/ja9050857.

An autoinhibited coiled-coil design strategy for split-protein protease sensors.

Author information

1
Department of Chemistry & Biochemistry, University of Arizona, Tucson, Arizona 85721, USA.

Abstract

Proteases are widely studied as they are integral players in cell-cycle control and apoptosis. We report a new approach for the design of a family of genetically encoded turn-on protease biosensors. In our design, an autoinhibited coiled-coil switch is turned on upon proteolytic cleavage, which results in the complementation of split-protein reporters. Utilizing this new autoinhibition design paradigm, we present the rational construction and optimization of three generations of protease biosensors, with the final design providing a 1000-fold increase in bioluminescent signal upon addition of the TEV protease. We demonstrate the generality of the approach utilizing two different split-protein reporters, firefly luciferase and beta-lactamase, while also testing our design in the context of a therapeutically relevant protease, caspase-3. Finally, we present a dual protease sensor geometry that allows for the use of these turn-on sensors as potential AND logic gates. Thus, these studies potentially provide a new method for the design and implementation of genetically encoded turn-on protease sensors while also providing a general autoinhibited coiled-coil strategy for controlling the activity of fragmented proteins.

PMID:
19803505
PMCID:
PMC2783329
DOI:
10.1021/ja9050857
[Indexed for MEDLINE]
Free PMC Article

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