Isothermal phase separation of protease-responsive elastin-like polypeptides

Elastin-like polypeptides (ELPs) have drawn interest as "smart" biopolymers due to their lower critical solution temperature (LCST) phase behavior. The LCST is sequence dependent: increasing hydrophobicity and molecular weight typically decreases the LCST. Sequence-defined phase behavior of ELPs has enabled their use in protein purification, therapeutic delivery, and injectable gels. However, temperature is not always a convenient trigger, especially in biological environments that maintain near-constant temperature, pH, and ionic composition. To expand ELP function, we demonstrate isothermal phase separation upon proteolytic cleavage of a diblock ELP. The ELP contains a protease recognition site between hydrophilic and hydrophobic blocks; the diblock sequence is designed for solubility in water at physiological temperature (37 °C). Incubation with a protease at 37 °C leads to cleavage of the diblock into soluble hydrophilic and insoluble hydrophobic blocks. We observe increases in turbidity attributed to spontaneous phase separation of the hydrophobic ELP. ELP designs are generalized by inserting recognition site for proteases involved in blood clotting or digestion. Protease-responsive ELPs provide an opportunity to exploit isothermal, biological triggers to tune biopolymer phase behavior and self-assembly processes.