Engineering Strategy and Vector Library for the Rapid Generation of Modular Light-Controlled Protein–Protein Interactions

Alexandra Madelaine Tichy, Elliot J. Gerrard, Julien M.D. Legrand, Robin M. Hobbs, Harald Janovjak

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)

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
Externally publishedYes

Bibliographical note


Funding Information:
We thank M. De Seram for assistance with experiments; E. Gschaider-Reichhart and S. Kainrath for discussions; and C. Tucker and C. Voigt for LSD genes. We acknowledge the facilities, scientific and technical assistance of Micromon and FlowCore at Monash University. The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government. The EMBL Australia Partnership Laboratory (EMBL Australia) is supported by the National Collaborative Research Infrastructure Strategy of the Australian Government. Elliot J. Gerrard was funded through the CSIRO Synthetic Biology Future Science Platform. Competing Interests: The authors report no conflict of interest. Preprint: http://www.biorxiv.org/content/10.1101/583369v1.

Publisher Copyright:
© 2019 Elsevier Ltd

Keywords

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

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