Advantages of substituting bioluminescence for fluorescence in a resonance energy transfer-based periplasmic binding protein biosensor

Abstract A genetically encoded maltose biosensor was constructed, comprising maltose binding protein (MBP) flanked by a green fluorescent protein (GFP 2 ) at the N-terminus and a Renilla luciferase variant (RLuc2) at the C-terminus. This Bioluminescence resonance energy transfer 2 (BRET 2 ) system showed a 30% increase in the BRET ratio upon maltose binding, compared with a 10% increase with an equivalent fluorescence resonance energy transfer (FRET) biosensor. BRET 2 provides a better matched Forster distance to the known separation of the N and C termini of MBP than FRET. The sensor responded to maltose and maltotriose and the response was completely abolished by introduction of a single point mutation in the BRET 2 tagged MBP protein. The half maximal effective concentration (EC 50 ) was 0.37 μM for maltose and the response was linear over almost three log units ranging from 10 nM to 3.16 μM maltose for the BRET 2 system compared to an EC 50 of 2.3 μM and a linear response ranging from 0.3 μM to 21.1 μM for the equivalent FRET-based biosensor. The biosensor’s estimate of maltose in beer matched that of a commercial enzyme-linked assay but was quicker and more precise, demonstrating its applicability to real-world samples. A similar BRET 2 -based transduction scheme approach would likely be applicable to other binding proteins that have a “venus-fly-trap” mechanism.