Complex coacervation of proteins and protein mixtures

Oppositely charged polyelectrolytes can undergo associative liquid-liquid phase separation, known as complex coacervation. This phase transition is driven by electrostatic attraction between the polymers and entropic gains from counterion release. An interesting class of complex coacervates relies on a protein as one of the charged polyelectrolytes. These hybrid protein-polymer coacervates demonstrate distinct behavior compared to polymer-polymer counterparts, largely due to the amphoteric and globular nature of the protein polyelectrolyte. Yet, the dense, protein-rich phase that forms upon phase separation has a range of potential applications from protein stabilization or purification and biomimetic compartments. Using techniques from protein engineering, we have designed protein libraries with varying charge and charge distribution to evaluate how these key parameters impact complex coacervation between globular proteins and linear polyelectrolytes. With this basic understanding, we have extended these efforts to more complex mixtures of proteins and polyelectrolytes. Using spectrally separated fluorescent proteins, we have quantitatively evaluated the phase separation of individual proteins as well as mixtures of these proteins with a model polycation. We have demonstrated that by tuning the salt concentration and pH, proteins of varying net charge can be selectively enriched and recovered from the complex mixture.