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

Sujan S. Shekhawat, Jason R. Porter, Akshay Sriprasad, Indraneel Ghosh

Research output: Contribution to journalArticle

54 Scopus citations

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 β-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.

Original languageEnglish (US)
Pages (from-to)15284-15290
Number of pages7
JournalJournal of the American Chemical Society
Volume131
Issue number42
DOIs
StatePublished - Oct 28 2009

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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