Skip to main content
. 2004 Oct 1;101(41):14701–14706. doi: 10.1073/pnas.0404107101

Fig. 1.

Fig. 1.

CFP/YFP-based sensors for detecting BoNT protease activity in vitro. (a) Schematic representation of the BoNT sensor constructs. CFP and YFP were connected via a fragment of synaptobrevin (amino acids 33–94, Upper), or SNAP-25 (amino acids 141–206, Lower), respectively. The cleavage sites for each BoNT are indicated. (b) Recombinant toxin sensor CFP-SNAP-25(141–206)-YFP yielded a FRET signal in vitro. His6-tagged CFP-SNAP-25(141–206)-YFP was selectively excited at 434 nm, which is optimal for CFP. The emission spectra are shown. The left arrow indicates the CFP emission peak; the right arrow indicates the YFP emission peak that is the result of FRET from the CFP donor. The emission spectra of recombinant his6-tagged CFP and YFP alone, as well as the mixture of these two proteins (1:1), are also shown. These control spectra indicate that YFP yields a small emission signal when directly excited at 434 nm, and that FRET does not occur unless CFP and YFP are linked together. (c) In vitro cleavage of toxin sensors monitored optically and via immunoblot analysis. (Upper) BoNT/A was mixed with CFP-SNAP-25(141–206)-YFP, and the emission spectra were collected at the indicated times after adding toxin. Cleavage of the sensor resulted in decreased FRET and a concurrent increase in the CFP fluorescence. (Lower) Samples (30 μl) were taken from the cuvette after each emission scan (in the experiment shown in Upper) and subjected to SDS/PAGE gel and immunoblot analysis. Cleavage of CFP-SNAP-25(141–206)-YFP was detected by using an anti-his6 antibody that recognizes the his6 tag at the N terminus of the sensor protein. (d) Sensor cleavage kinetics monitored by using a plate reader. CFP-SNAP-25(141–206)-YFP was cleaved with BoNT/A (100 pM) as detailed in Materials and Methods. CFP-SNAP-25(141–206)-YFP alone, as well as the CFP/YFP mixture with added BoNT/A, were analyzed in parallel as controls. (e) Picomolar sensitivity of the toxin sensors. (Left) Experiments were carried out as in d after measuring the emission ratio after 4 h; these values are plotted against the log of the toxin concentration. (Right) The EC50 values for BoNT/A, -E, -B, and -F, when incubated with toxin sensors for 4 or 16 h, were determined as described for Left. Each value represents the mean of three independent determinations.