Immobilization and enhancement of a heterodimeric fluorescent biosensor in fibrous protein biomaterials via gene fusion.

Abstract

Leave-One-Out Green Fluorescent Proteins (LOO_GFPs) unite a peptide sensor and detection method in a single biocompatible molecule. In LOO_GFPs, one β-strand of the GFP protein is removed and the cavity is re-engineered to specifically bind a desired analyte. Unfortunately, LOO_GFPs have a reduced quantum yield relative to the parent protein, and form fluorescent oligomers in the unbound state, reducing signal-to-noise. Immobilizing LOO_GFPs in materials composed of the Drosophila protein Ultrabithorax (Ubx) via gene fusion both increased the fluorescent signal and prevented oligomerization, substantially reducing background noise. These fibers represent the first incorporation of aheterodimeric protein into materials via gene fusion. Interactions between LOO_GFP and Ubx that hampered analyte rebinding were mitigated by optimizing salt and detergent concentrations in the assay. The result is a useful first-generation fluorescent biosensor, immobilized in and stabilized by robust protein fibers. This study highlights the advantages and identifies potential pitfalls associated with protein immobilization in materials.