A number of intrinsically-disordered proteins (IDPs) assemble via phase separation into spherical liquid droplets. These droplets can 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, the RGG domain from LAF-1, and produced a variant consisting of two RGG domains in tandem, separated by a thrombin cleavage site. This tandem RGG protein exhibits phase separation as a function of protein concentration, temperature, and salt concentration. Importantly, assembly of tandem RGG droplets occurs under physiological conditions at lower protein concentration than is observed with a single RGG domain. Thus, operating at a protein concentration at which tandem RGG phase separates but RGG does not, thrombin treatment causes droplets formed by tandem RGG to slowly dissolve. The kinetics of dissolution were measured by quantitative optical microscopy and fit to a reaction-diffusion model. We next demonstrated two strategies to release passenger proteins from the microspheres. In the first approach, the passenger protein is incorporated into tandem RGG as a genetic fusion, and the passenger protein 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. This receptor-ligand interaction recruits the passenger protein into the droplets, and protease treatment to cleave off the ligand de-recruits the passenger protein from the droplets. We anticipate that these recombinant materials could 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 from microspheres. Furthermore, we envision applications in engineering the behavior of membrane-less organelles within cells.

Protease-responsive microspheres engineered from self-assembled disordered proteins

254th National Meeting (2017)

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