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 (GFP2) at the N-terminus and a Renilla luciferase variant (RLuc2) at the C-terminus. This Bioluminescence resonance energy transfer2 (BRET2) 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. BRET2 provides a better matched Förster 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 BRET2 tagged MBP protein. The half maximal effective concentration (EC50) was 0.37μM for maltose and the response was linear over almost three log units ranging from 10nM to 3.16μM maltose for the BRET2 system compared to an EC50 of 2.3μM and a linear response ranging from 0.3μM to 21.1μM 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 BRET2-based transduction scheme approach would likely be applicable to other binding proteins that have a “venus-fly-trap” mechanism.