Fluorescent protein-based biosensors would have considerable application in cell biology, proteomics, and immunochemistry. To this end, the widely used green fluorescent protein (GFP) allows for easy detection, but attempts to graft multiple binding loops onto GFP to impart affinity for a specific target have been met with limited success due to the structural instability of the GFP chromophore. In this study, using a surrogate loop approach and directed evolution by yeast surface display, GFP scaffolds capable of accommodating two random loops were created. Initially, two model binding loops were grafted onto the GFP scaffold so as to form a putative binding interface. The resulting protein was not expressed by yeast nor was it fluorescent. We therefore employed directed evolution to engineer the loop-inserted GFP scaffold to be well expressed and fluorescent. To bias directed evolution towards scaffolds that fold correctly and have stable chromophores, selection pressures of elevated expression temperature (37°C) and thermal stability (up to 70°C) were employed. Using the resultant scaffolds, the inserted amino acids in both grafted loop regions were randomized to create a library of potential biosensors. The library was successfully screened to identify GFP-based binders to several antigens, and the GFP-based binders were produced and validated in a variety of biological assays including flow cytometry and immunocytochemistry. Through this work, novel insights into the engineering of intrinsically fluorescent proteins have been gained leading to a new biosensor platform.

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