(596f) Engineering pH Sensitive Fusion Protein Vesicles

Protein vesicles can be synthesized by mixing two fusion proteins; an elastin-like polypeptide (ELP) fused to an arginine rich leucine zipper (Z_R) with a globular, hydrophilic protein fused to a glutamate rich leucine zipper (Z_E). The ELP serves as the hydrophobic block and drives vesicle self-assembly due to a hydrophobic transition that occurs as ELP is heated. Elastin-like polypeptides are repeats of the amino acids VPGXG where X can be any amino acid except proline. Incorporating histidine into the guest residue of the ELP has been shown to make ELP pH sensitive and results in the reversal of the hydrophobic transition when the pH is reduced below 7.0. Incorporating pH sensitivity into fusion protein vesicles would allow for pH dependent disassembly of vesicles and release of entrapped molecules. In this work, three His-Z_R-ELP fusion proteins with 20, 40, and 60 percent histidine in the ELP guest positions were created and studied to determine their pH dependent assembly and disassembly properties. To demonstrate the formation of pH responsive vesicles, the red fluorescent fusion protein mCherry-Z_E was used as a model globular, hydrophilic domain. The transition temperature of His-Z_R-ELP/ mCherry-Z_E mixtures was measured at pH’s between 5.5 to 8.0, which revealed that the vesicles are pH responsive and the histidine content in His-Z_R-ELP affects self-assembly. The transition temperature increases as the pH of the solution decreases and the increase in transition temperature was more drastic for His-Z_R-ELP variants with higher histidine content. Additionally, these mixtures were characterized using dynamic light scattering, small angle neutron scattering, and fluorescent microscopy to show that reducing the pH of the protein mixture prior to temperature induced vesicle assembly results in the formation of larger . Vesicles formed at neutral or basic pH swelled upon addition of acid, releasing encapsulated cargos. These pH sensitive vesicles have the potential to be used in biocatalytic or drug delivery applications where pH dependent release could enable separations or be utilized for drug release in acidic tissues or cellular compartments.