(649a) Protease-Responsive Droplets Engineered from Self-Assembled Disordered Proteins

Certain intrinsically-disordered proteins (IDPs) self-assemble via liquid-liquid phase separation into spherical droplets. These droplets have been discovered to function as membrane-less organelles in living cells. Here, we engineered protease-responsive microspherical droplets from self-assembling recombinant IDPs. We selected a prototypical droplet-forming IDP domain, the arginine- and glycine-rich RGG domain from LAF-1, and designed a variant consisting of two RGG domains in tandem, separated by a thrombin protease cleavage site. This variant, which we refer to as tandem RGG, exhibits phase separation as a function of protein concentration, temperature, and salt concentration. Importantly, due to multivalent interactions, assembly of tandem RGG droplets occurs under physiological conditions at lower protein concentration than is observed with a single RGG domain. We harnessed this property to engineer protease-triggered droplet dissolution. By operating at a protein concentration at which tandem RGG phase separates but a single RGG domain does not, thrombin treatment causes droplets formed by tandem RGG to slowly dissolve, simply by cleaving tandem RGG in half. We measured the kinetics of droplet dissolution by quantitative optical microscopy and interpreted the results with a reaction-diffusion model. Next, we demonstrated two strategies to release passenger proteins from the droplets. In the first approach, the passenger protein is incorporated into tandem RGG as a genetic fusion and is released as the droplets dissolve upon thrombin treatment. In the second approach, tandem RGG is tagged with a receptor and the passenger protein is tagged with a high-affinity ligand to that receptor. This receptor-ligand interaction recruits the passenger protein into the droplets; subsequent protease treatment to cleave off the ligand de-recruits the passenger protein from the droplets, while the droplets remain intact. We anticipate that these recombinant biomolecular materials will have applications as a technology for topical drug delivery to wounds, where thrombin is present, and more broadly as a modular platform for controlled release of peptides and proteins. We also envision applications in engineering the behavior of membrane-less organelles within cells.