(326b) Development of a Universal Protein Switch Platform Using 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).    In these protein switches, BLA catalytic activity is a function of maltose concentration.  Creating new protein switches that are activated by different ligands requires the availability of the prerequisite ligand-binding domains (the input domain of the switch).  The process of developing a new switch is also time consuming and labor intensive.  To accelerate our ability to construct switches that are activated by new protein and small molecule targets, we have developed protein switches using monobody and DARPin antibody mimetics as input domains.   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 simply by transplanting mutations known to cause binding to new targets in the antibody mimetic domain alone.  These results outline a general strategy for the more rapid creation of protein switches.