ENZYME ASSAY WITH DUPLICATE FLUOROPHORES

摘要

Compositions and methods are disclosed that provide a rapid, sensitive, and accurate cell-based assay for enzyme activity, particularly for enzyme activities associated with botulinum toxins. A cell is provided that expresses a construct that includes an anchor region, a cleavage site, and a reporting region having two or more identical reporter peptides. Enzymatic activity at the cleavage site releases the reporter region into the cytosol, where multiple degradation events occur. The observed change in the signal is proportional to the enzymatic activity.

主权项

1. A reporting construct for characterizing an enzyme activity comprising:
a membrane anchoring domain comprising a first peptide that forms a complex with a membrane of a cell; a reporter domain comprising first occurrence of a second peptide that produces a first signal at a first wavelength and a second occurrence of the second peptide that produces a second signal at the first wavelength, wherein the reporter domain produces an aggregate signal that is a summation of the first signal and the second signal; a cleavage site comprising a third peptide, the cleavage site interposed between the membrane anchoring domain and at least two of the two or more occurrences of the reporter domain, wherein the third peptide is selected to undergo a cleavage event upon exposure to the enzyme activity, wherein the aggregate signal is observable in the absence of either one of the first signal or the second signal.