Isothermal phase separation of cleavable elastin-like polypeptides

Elastin-like polypeptides (ELPs) have drawn interest as thermally responsive biopolymers due to their lower critical solution temperature (LCST) phase behavior. ELPs are soluble in water at temperatures below the LCST and undergo reversible phase separation upon heating above the LCST. The LCST is sequence dependent: increasing hydrophobicity and molecular weight typically decreases the LCST. Sequence-defined, thermo-responsive behavior has enabled the use of ELPs for 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 cleavage of a diblock ELP. The diblock ELP contains a protease recognition site between hydrophilic and hydrophobic blocks; the diblock sequence is designed for solubility in water at 37 °C. Incubation with a protease at 37 °C leads to cleavage of the diblock ELP into a hydrophilic ELP (LCST > 37 °C, soluble) and a hydrophobic ELP (LCST < 37 °C, insoluble). We observe an increase in turbidity, which is attributed to spontaneous phase separation of the hydrophobic ELP. Overall, cleavable ELPs provide an opportunity to exploit isothermal, biological triggers for phase behavior and self-assembly processes.