Two-component protein hydrogels assembled using an engineered disulfide-forming protein-ligand pair.

We present the development of a two-component self-assembling protein hydrogel. The building blocks of the hydrogel are two liquid-phase protein block copolymers each containing (1) a subunit of the trimeric protein CutA as a cross-linker and (2) one member of a PDZ-domain-containing protein-ligand pair whose interaction was reinforced by an engineered disulfide linkage. Mixing of the two building blocks reconstitutes a self-assembling polypeptide unit, triggering hydrogel formation. This hydrogel exhibits extremely high solution stability at neutral and acidic pHs and in a wide range of temperatures (4-50 °C). Incorporation of a "docking station peptide" binding motif into a hydrogel building block enables functionalization of the hydrogel with target proteins tagged with a "docking protein". We demonstrated the application of an enzyme-functionalized hydrogel in a direct electron transfer enzymatic biocathode. These disulfide-reinforced protein hydrogels provide a potential new material for diverse applications including industrial biocatalysis, biosynthesis, biofuels, tissue engineering, and controlled drug delivery.