Controlled Liquid-Liquid Phase Seperation of Recombinant Oleosin

Abstract

Recombinant proteins enable the design of materials with tailored functionality and responsiveness. We designed a protein that, upon oxidation of a cysteine residue, phase separates into micron sized liquid droplets. These droplets are similar to membrane-less organelles found in cells. This work is based on the natural plant protein, oleosin. Oleosin is an amphiphile with distinct hydrophilic and hydrophobic domains: N and C-terminal hydrophilic segments and a hydrophobic central core. From wild type oleosin, a truncated version was engineered that reduced the hydrophobic core and added glycines. An amino acid was then mutated to add a cysteine in the N-terminal hydrophilic segment. Above an upper critical solution temperature, the protein formed micelles, and below this temperature, the protein condensed to form liquid droplets. The phase transition was thermoreversible. The cysteine containing protein had a higher propensity to form droplets than the variant with no cysteines. We predict that this is because disulfide bonding of the cysteine residues created dimers. In other systems, dimers have been shown to have a higher propensity to form droplets. A family of oleosins was synthesized with a single cysteine at various locations in the protein backbone. Placing the cysteine closer to the N-terminus resulted in a higher transition temperature. We predict that this is because cysteine residues closer to the N-terminus are exposed on the protein surface and therefore oxidizes more readily. This protein construct provides a novel way to control protein liquid droplet formation and dissolution. We envision this work having applications as a membrane-less organelle mimic in synthetic protocells and as a targeted drug delivery system.