Modular Ligand-Activated Enzymes from Antibody Mimetic Proteins

Ligand-activated enzymes (protein switches) can be constructed by the correct fusion of ligand-binding domains and enzymes.   For example, maltose-activated beta-lactamases have been constructed by the insertion of beta-lactamase (BLA) into the maltose binding protein (MBP) from E. coli.    In these protein switches, BLA catalytic activity is a function of maltose concentration.  The process of developing a new switch is time consuming and labor intensive. To accelerate our ability to construct switches that are activated by new protein and small molecule targets, we have worked to develop modular platforms for protein switches using monobody and DARPin antibody mimetics as input domains. The advantage of these proteins is that they have been engineered to bind with high affinity to wide variety of protein targets. In contrast to the periplasmic binding proteins, however, these antibody mimetic proteins do not undergo a large conformational change on ligand binding. First, MBP-activated beta-lactamases were created by a directed evolution approach involving the fusion of BLA with the MBP-binding DARPin off7 and the fusion of BLA with the MBP-binding monobody MBP-74.  We identified switches that function both in vitro and in E. coli cells.  Next, these MBP-activated switches were converted for activation by new protein targets from bacteria, yeast and other eukaryotes by transplanting mutations into the antibody mimetic domain known to cause binding to new targets. The modularity of these protein switches will be discussed along with the implications for their future use as potential biosensors.