(284d) Cononsolvency of the Elastin-like Polypeptide in Binary Aqueous Solutions and Its Application to Protein Purification Processes

Elastin-like polypeptides (ELPs) are a family of biopolymers characterized by the VPGXG repeat unit motif, where X can be any amino acid except proline. One unique feature of these ELPs is their lower critical solution transition (LCST) behavior in aqueous solutions, in which the polypeptide is soluble at low temperatures and insoluble at high temperatures. This LCST behavior is key to many of the promising applications for ELP-based materials. For example, it is this LCST behavior that enables formulation of ELP-based injectable materials that exhibit controlled dynamic release of therapeutic molecules. This LCST behavior is also the fundamental basis of inverse thermal cycling (ITC) purification of ELP-tagged proteins. Thus, the ability to tune the ELP transition temperature is key to many applications of interest. The phase behavior of these ELPs has been thoroughly explored in the context of both ELP sequence and solution salt content. However, little is known about the behavior of these ELPs in binary aqueous solutions, including whether they exhibit cononsolvency similar to that observed in synthetic polymers such as poly(N-isopropyl acrylamide) (PNIPAM). Here, we characterize the phase behavior of ELPs in water-ethanol blends across various sodium chloride concentrations. Phase transition temperatures of ELPs are measured as a function of both solvent and sodium chloride content using turbidimetry.

These experiments reveal the existence of both lower critical solution transition (LCST) and upper critical solution transition (UCST) like behavior of ELPs in certain solvent blends that also produce LCST and UCST behavior in the synthetic polymer, PNIPAM, constituting the first known reports of ELP cononsolvency. It is found that sodium chloride, previously shown to suppress the LCST of ELPs in aqueous solution, has non-monotonic effects on the transition temperature of ELPs in this cononsolvent regime, with ~10-75 mM NaCl decreasing the UCST-like transition temperature, and higher salt concentrations returning the UCST-like transition temperature to its 0 mM NaCl value. Finally, we demonstrate the usefulness of this cononsolvency in protein purification processes on the ELP-green fluorescent protein (GFP) fusion protein system. Here, the cononsolvency phenomenon is used to selectively precipitate the ELP-GFP fusion protein in a process analogous to inverse thermal cycling (ITC) of ELP-tagged fusion proteins. Importantly, it is shown that solvent-induced protein precipitation leads to significantly lower levels of residual salt in the final product than traditional ITC, demonstrating the potential to eliminate the need for dialysis in protein purification using this strategy. Furthermore, this work greatly expands the handles available to tune ELP transition behavior.