Protease-Driven Phase Separation of Elastin-Like Polypeptides

Elastin-like polypeptides (ELPs) are a promising material platform for engineering stimuli-responsive biomaterials, as ELPs undergo phase separation above a tunable transition temperature. ELPs with phase behavior that is isothermally regulated by biological stimuli remain attractive for applications in biological systems. Herein, we report protease-driven phase separation of ELPs. Protease-responsive "cleavable" ELPs comprise a hydrophobic ELP block connected to a hydrophilic ELP block by a protease cleavage site linker. The hydrophilic ELP block acts as a solubility tag for the hydrophobic ELP block, creating a temperature window in which the cleavable ELP reactant is soluble and the proteolytically generated hydrophobic ELP block is insoluble. Within this temperature window, isothermal, protease-driven phase separation occurs when a critical concentration of hydrophobic cleavage product accumulates. Furthermore, protease-driven phase separation is generalizable to four compatible protease-cleavable ELP pairings. This work presents exciting opportunities to regulate ELP phase behavior in biological systems using proteases.