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

Publication date: Available online 29 May 2019Source: Journal of Molecular BiologyAuthor(s): Alexandra-Madelaine Tichy, Elliot J. Gerrard, Julien M.D. Legrand, Robin M. Hobbs, Harald JanovjakAbstractOptogenetics enables the spatio-temporally precise control of cell and animal behaviour. 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.Graphical abstract
Source: Journal of Molecular Biology - Category: Molecular Biology Source Type: research