Creation of GFAbs: GFP-based biosensors possessing the binding properties of antibodies

Traditional protein detection assays such as Western blots, ELISAs, flow cytometry, and immunocytochemistry have been performed by utilizing antibodies as protein-based biosensors. However, antibodies are expensive to produce and detection of the binding of the antibody to the protein target requires chemical conjugation of a fluorescent probe to the antibody or some secondary means of detection. A protein-based biosensor that would be superior to an antibody is one that, like antibodies, binds to its molecular target with high affinity and specificity, but also possesses an inherent property that is easy to detect, and is inexpensive to produce. Green fluorescent protein (GFP) has the intrinsic property of fluorescence thus allowing for its easy detection. However, to convert GFP into a protein-based biosensor, one would need to endow it with binding properties by inserting binding loops into GFP such that these binding loops confer affinity and specificity for the target. Moreover, a very diverse collection of such binding loop inserted-GFPs with each GFP having a different set of binding loops could in principle faciliate rapid isolation of a GFP-based biosensor against any target of interest.;To achieve this objective, we utilized yeast surface display to study the ability of yeast codon optimized enhanced GFP to accommodate surrogate binding loops inserted in two solvent exposed loop regions of GFP that are in close proximity to each other. GFP was unable to retain fluorescence and expression upon dual binding loop insertion. We chose to engineer the GFP scaffold such that the scaffold can accommodate any dual binding loop insertions. We have created a set of GFP scaffolds that are fluorescent and well expressed while displaying dual surrogate binding loops. Further, we have created a library of random dual loop inserted GFPs using this set of scaffolds and utilized the library to isolate GFP-based biosensors or GFAbs that bind to protein targets with high affinity (single digit nanomolar binding affinity) and specificity. Finally, we have demonstrated that we can produce these GFP-based biosensors as fluorescent binding proteins.