Engineering strategy and vector library for the rapid generation of modular light-controlled protein–protein interactions

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Abstract

Optogenetics enables the spatio-temporally precise control of cell and animal behavior. Many optogenetic tools are driven by light-controlled protein–protein interactions (PPIs) that are repurposed from natural light-sensitive domains (LSDs). Applying light-controlled PPIs to new target proteins is challenging because it is difficult to predict which of the many available LSDs, if any, will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of light-controlled PPIs. The strategy permits fusing a target protein to multiple LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs, many of which have been previously applied ex vivo and in vivo. We demonstrate the versatility of the approach and the necessity for sampling LSDs by generating light-activated caspase-9 (casp9) enzymes. Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology.

Original languageEnglish
Pages (from-to)3046-3055
Number of pages10
JournalJournal of Molecular Biology
Volume431
Issue number17
DOIs
Publication statusPublished - 9 Aug 2019

Keywords

  • Optogenetics
  • light-sensitive domain
  • photoreceptor
  • protein-protein interaction
  • caspase9

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