(272c) Protease-Driven Phase Separation of Elastin-like Polypeptides

Elastin-like polypeptides (ELPs) are promising biomaterials for medical applications owing to their biocompatibility and tunable phase behavior; however, viable stimuli to trigger phase separation in vivo remain limited. Temperature remains the predominant stimulus for promoting ELP phase separation, as ELPs become insoluble and aggregate above a sequence-dependent transition temperature. In therapeutic contexts, the relatively constant human body temperature poses a significant challenge to exploiting temperature-driven ELP phase separation. To address inherent limitations of temperature as an in vivo stimulus, we demonstrate isothermal ELP phase separation triggered by a protease.

The designed protease-responsive “cleavable” ELP was soluble at physiologically relevant temperatures and produced an insoluble ELP fragment upon incubation with a protease. The cleavable ELP contains a tobacco etch virus (TEV) protease cleavage site between hydrophilic and hydrophobic ELP blocks, where hydrophilicity of each block was tuned by altering their amino acid compositions. Initially, the cleavable ELP was soluble in aqueous environments, with the hydrophilic block acting as a solubility tag for the hydrophobic block. Upon incubation at 37 °C with TEV protease, we observed an increase in turbidity attributed to phase separation of the hydrophobic ELP block following proteolytic removal of the hydrophilic ELP block. Reaction temperature and concentrations amenable to protease-driven phase separation were predicted with a phase diagram of the cleavable ELP and its hydrophobic cleavage product. Protease-driven phase separation generalized to diverse proteases by changing the protease cleavage site in the cleavable ELP. In conclusion, we demonstrated isothermal ELP phase separation driven by proteolytic cleavage of a soluble ELP, thereby creating opportunities to exploit solely biological triggers for driving ELP phase separation.