A Chimeric Fusion Protein Engineered with Disparate Functionalities—Enzymatic Activity and Self-assembly

The fusion of protein domains is an important mechanism in molecular evolution and a valuable strategy for protein engineering. We are interested in creating fusion proteins containing both globular and structural domains so that the final chimeric protein can be utilized to create novel bioactive biomaterials. Interactions between fused domains can be desirable in some fusion protein applications, but in this case the optimal configuration will enable the bioactivity to be unaffected by the structural cross-linking. To explore this concept, we have created a fusion consisting of a thermostable aldo-keto reductase, two α-helical leucine zipper domains, and a randomly coiled domain. The resulting protein is bifunctional in that (1) it can self-assemble into a hydrogel material as the terminal leucine zipper domains form interprotein coiled-coil cross-links, and (2) it expresses alcohol dehydrogenase and aldo-keto reductase activity native to AdhD from Pyrococcus furiosus. The kinetic parameters of the enzyme are minimally affected by the addition of the helical appendages, and rheological studies demonstrate that a supramolecular assembly of the bifunctional protein building blocks forms a hydrogel. An active hydrogel is produced at temperatures up to 60 °C, and we demonstrate the functionality of the biomaterial by monitoring the oxidation and reduction of the native substrates by the gel. The design of chimeric fusion proteins with both globular and structural domains is an important advancement for the creation of bioactive biomaterials for biotechnology applications such as tissue engineering, bioelectrocatalysis, and biosensing and for the study of native assembled enzyme structures and clustered enzyme systems such as metabolons.