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

Autor: Tichy AM; Australian Regenerative Medicine Institute (ARMI), Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton/Melbourne, VIC 3800, Australia; European Molecular Biology Laboratory Australia (EMBL Australia), Monash University, Clayton/Melbourne, VIC 3800, Australia; Institute of Science and Technology Austria (IST Austria), 3400 Klosterneuburg, Austria., Gerrard EJ; Australian Regenerative Medicine Institute (ARMI), Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton/Melbourne, VIC 3800, Australia; European Molecular Biology Laboratory Australia (EMBL Australia), Monash University, Clayton/Melbourne, VIC 3800, Australia; Commonwealth Scientific and Industrial Research Organisation, Synthetic Biology Future Science Platform, Monash University, Clayton/Melbourne, VIC 3800, Australia., Legrand JMD; Australian Regenerative Medicine Institute (ARMI), Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton/Melbourne, VIC 3800, Australia; Development and Stem Cells Program, Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Clayton/Melbourne, VIC 3800, Australia., Hobbs RM; Australian Regenerative Medicine Institute (ARMI), Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton/Melbourne, VIC 3800, Australia; Development and Stem Cells Program, Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Clayton/Melbourne, VIC 3800, Australia., Janovjak H; Australian Regenerative Medicine Institute (ARMI), Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton/Melbourne, VIC 3800, Australia; European Molecular Biology Laboratory Australia (EMBL Australia), Monash University, Clayton/Melbourne, VIC 3800, Australia; Institute of Science and Technology Austria (IST Austria), 3400 Klosterneuburg, Austria. Electronic address: harald.janovjak@monash.edu.
Jazyk: angličtina
Zdroj: Journal of molecular biology [J Mol Biol] 2019 Aug 09; Vol. 431 (17), pp. 3046-3055. Date of Electronic Publication: 2019 May 29.
DOI: 10.1016/j.jmb.2019.05.033
Abstrakt: 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.
(Copyright © 2019 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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