Transforming protein-polymer conjugate purification by tuning protein solubility*

Almost all commercial therapeutic and industrial proteins are purified by processes that include salting-out precipitation in ammonium sulfate. Protein-polymer conjugates are generally synthesized from already pure starting materials and the struggle to separate the conjugates from polymer, native protein, and from differently modified variants has vexed scientists for decades. Since ammonium sulfate precipitation is exclusively used as an initial step in crude protein purifications, it has had little relevance in the delicate purification of protein-polymer conjugates. We have discovered, however, that polymers grown from the surface of proteins have a transformational effect on the solubility of proteins in salt solutions. We generated a family of protein-polymer conjugates with a variety of polymers, grafting densities, and polymer lengths using surface-initiated atom transfer radical polymerization. Covalently attached polymers increased solubility of the conjugates in ammonium sulfate and completely prevented precipitation. Molecular dynamic simulations showed the impact was driven by an anti-polyelectrolyte effect. We then efficiently and simply purified mixtures of conjugates and native proteins.