Split enzymes: Design principles and strategy

Engineering precise control of enzymatic activity provides a powerful means to manipulate and understand biological processes. One approach to achieve a switch-like control over enzyme activity is to design a split enzyme, where the protein is separated into two polypeptides with each inactive fragment fused to inducible dimerization domains. The activity of the enzyme can be controlled by the addition of a small molecule, which causes the inducible dimerization domains to come together and reconstitute the split enzyme and its activity. In recent years, split enzymes have been designed for a variety of enzyme classes, and these synthetic molecular tools have enabled spatial and temporal dissection of biological processes in ways that were difficult previously. Here, we summarize key design principles and strategies to guide future split enzyme engineering efforts, using split enzymes generated from our research group as examples.