Advantages of substituting bioluminescence for fluorescence in a resonance energy transfer-based periplasmic binding protein biosensor.
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 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 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 10nM 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.
CSIRO Food Futures National Research Flagship & Ecosystem Sciences, Australia, Canberra ACT 2601, Australia. Helen.dacres@csiro.au
Helen
H
Dacres
Michelle
M
Michie
Alisha
A
Anderson
Stephen C
SC
Trowell
D001665:Binding Sites; D015374:Biosensing Techniques / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation*; D008163:Luminescent Measurements / Q000295:instrumentation*; D008320:Maltose / Q000737:chemistry; D058619:Maltose-Binding Proteins / Q000032:analysis* / Q000737:chemistry*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D058619:Maltose-Binding Proteins; D008320:Maltose