Engineering of functional proteinaceous hydrogels for biotechnology applications

Hydrogels consisting of 3-D cross-linked polymer networks have emerged as interesting scaffolds for a host of applications including drug delivery, tissue engineering, bioelectronics and biosensors. We have used protein engineering to create hydrogels by genetically appending cross-linking domains to globular proteins, which results in proteinacous biomaterials that exhibit bioactivity. Previously, we have investigated α-helical leucine zipper appendages for cross-linking. By genetically fusing these helices to the termini of globular proteins, we have created hydrogels with fluorescent proteins, and more interestingly, with enzymes such as laccase, organophosphate hydrolase (OPH) and alcohol dehydrogenase (AdhD). More recently, we have created a hydrogel from three self-assembling dehygrogenases which results in a biomaterial that is also a synthetic metabolic pathway capable of oxidizing methanol to CO2. Now we are exploring the calcium-responsive repeats-in-toxin (RTX) domain as an alternative cross-linker. In the absence of calcium, the RTX domain is unstructured. In calcium rich environments, it reversibly folds into a beta roll structure. By engineering one face of the beta roll to contain leucines, we created an environmentally-responsive cross-linking domain capable of calcium-triggered self-assembly. The folded beta roll contains a second face, which we have also designed to contain leucines. We are currently characterizing this “double-face” leucine-rich RTX domain and evaluating is mechanical and biophysical properties. The RTX domains represent a truly “smart” cross-linking strategy in that the addition of a specific effector molecule (calcium) enables the reversible formation of the hydrogel network.