Complexation of proteins with polyelectrolytes or block copolymers can lead to phase separation to generate a coacervate phase or self-assembly of coacervate core micelles. However, many proteins do not coacervate at conditions near neutral pH and physiological ionic strength. Here, protein supercharging is used to systematically explore the effect of protein charge on the complex coacervation with polycations. Four model proteins were anionically supercharged to varying degrees as quantified by mass spectrometry. Proteins phase separated with strong polycations when the ratio of negatively charged residues to positively charged residues on the protein (α) was greater than 1.1–1.2. Efficient partitioning of the protein into the coacervate phase required larger α (1.5–2.0). The preferred charge ratio for coacervation was shifted away from charge symmetry for three of the four model proteins and indicated an excess of positive charge in the coacervate phase. The composition of protein and polymer in the coacervate phase was determined using fluorescently labeled components, revealing that several of the coacervates likely have both induced charging and a macromolecular charge imbalance. The model proteins were also encapsulated in complex coacervate core micelles and micelles formed when the protein charge ratio α was greater than 1.3–1.4. Small angle neutron scattering and transmission electron microscopy showed that the micelles were spherical. The stability of the coacervate phase in both the bulk and micelles improved to increased ionic strength as the net charge on the protein increased. The micelles were also stable to dehydration and elevated temperatures.

The synthetic modification of proteins plays an important role in chemical biology and biomaterials science. These fields provide a constant need for chemical tools that can introduce new functionality in specific locations on protein surfaces. In this work, an oxidative strategy is demonstrated for the efficient modification of N-terminal residues on peptides and N-terminal proline residues on proteins. The strategy uses o-aminophenols or o-catechols that are oxidized to active coupling species in situ using potassium ferricyanide. Peptide screening results have revealed that many N-terminal amino acids can participate in this reaction, and that proline residues are particularly reactive. When applied to protein substrates, the reaction shows a stronger requirement for the proline group. Key advantages of the reaction include its fast second-order kinetics and ability to achieve site-selective modification in a single step using low concentrations of reagent. Although free cysteines are also modified by the coupling reaction, they can be protected through disulfide formation and then liberated after N-terminal coupling is complete. This allows access to doubly functionalized bioconjugates that can be difficult to access using other methods.

Polyelectrolytes of opposite charge in aqueous solution can undergo a liquid–liquid phase separation known as complex coacervation. Complex coacervation of ampholytic proteins with oppositely charged polyelectrolytes is of increasing interest as it results in a protein rich phase that has potential applications in protein therapeutics, protein purification, and biocatalysis. However, many globular proteins do not phase separate when mixed with an oppositely charged polyelectrolyte, and those that do phase separate do so over narrow concentration, pH, and ionic strength ranges. The protein design factors that govern complex coacervation under varying conditions are still relatively unexplored. Recent work indicates that proteins with an intrinsically disordered region, a higher net charge, or a patch of charged residues are more likely to undergo a phase transition. Based on these design parameters, polyionic coacervation tags were designed and assessed for their ability to promote protein complex coacervation with oppositely charged polyelectrolytes. The phase behavior of a panel of engineered proteins was evaluated with the strong polycation poly(4-vinyl N-methyl pyridinium iodide). Proteins containing the ionic tags formed liquid coacervate droplets, while isotropically charged protein variants formed solid precipitates. The ionic tags also promoted phase separation at higher salt concentrations than an isotropic distribution of charge on the protein surface. The salt dependence of the protein complex coacervation could be predicted independently for tagged or isotropic variants by the ratio of negative-to-positive residues on the proteins and universally by calculating the distance between like charges. The addition of just a six residue polyionic tag generated a globular protein capable of liquid–liquid phase separation at physiological pH and ionic strength. This model system has provided the initial demonstration that short, ionic polypeptide sequences (6–18 amino acids) can drive the liquid–liquid phase separation of globular proteins.