(593aj) Modular Protein Switches Employing Fibronectin-Derived Monobodies As Input Domains

Biological sensor platforms often combine a recognition element (e.g. an antibody) that binds to a target molecule and an output scheme to produce a readable signal. There is a need to develop biosensors with a simple, direct connection between target binding and output signal in a platform that is readily adaptable for the detection of any biological targets.  We have developed target-activated enzyme switches that employ a fibronectin type III-based synthetic binding domain (called a monobody) as the target recognition domain and TEM-1 β-lactamase (BLA) as the output domain. Monobodies have potential as antibody mimetics and have been engineered by others to specifically bind a variety of protein targets including maltose binding protein (MBP).

We implemented a novel, PCR-based combinatorial approach to create a library in which a circularly permuted β-lactamase was inserted at all possible locations in the monobody MBP-74 that specifically binds MBP.   This library was subjected to a bacterial band-pass genetic selection system to identify gene-fusions that confer increased resistance to β-lactam antibiotics when MBP is coexpressed.  A number of switch genes conferring this phenotype were identified, with the best genes conferring a 16-fold increase in β-lactam antibiotic resistance in the presence of MBP.   The switching activity resulted from a combination of allosteric effects (i.e. MBP allosterically activates the BLA domain’s enzyme activity) and cellular accumulation affects (i.e. the switch protein accumulates at higher levels in the presence of MBP). We tested the modularity of these switches by introducing mutations to the monobody domain that are known to convert their binding specificity to new target proteins such as yeast small ubiquitin-like modifier protein (ySUMO) and green fluorescent protein (GFP). The altered switches demonstrated both allosteric switching and the ability to confer a switching phenotype to cells.  These experiments demonstrate the versatility and modularity or monobody-based protein switches.