Functional analysis of small-molecule and protein binding domains using monomeric streptavidin-based two-component biosensors

Protein biosensors are important analytical and diagnostic tools that convert molecular recognition into measurable activity. Most common two-component biosensors require two non-competing recognition domains, yet binding affinity and specificity alone do not reliably predict their compatibility with the rest of the biosensor. We report a modular platform that exploits monomeric streptavidin (mSA2) as a surrogate recognition domain, enabling rapid functional evaluation of candidate binders in the context of biosensor scaffold without requiring a second ligand-specific binding protein. We established a chromatography-free protocol for recovering functional mSA2-based biosensor components from Escherichia coli. Using biotinylated small molecules methotrexate and vitamin D3, as well as heart failure protein biomarkers galectin-3 and S100A7 we demonstrate ligand-dependent reporter biosensor activation. The developed platform provides a scalable approach for rapid binder evaluation and biosensor prototyping, streamlining biosensor development using binding domains generated by directed evolution or computational protein design.