N-end rule protease activity indication methods and uses thereof
View Patent ↗A cell based assay for detection for protease activity is disclosed. In the assay a cell is engineered to express a protease substrate with at least one label, preferably on its C-terminus. Cleavage of the substrate by the protease that recognizes it results in a C-terminal fragment and a N-terminal fragment, where the fragment having the label is subject to ubiquitin proteasome degradation. The assay measures the disappearance of the label due to degradation of the fragment to which it is attached. A cell free assay is also described for detection of protease activity. In the cell free assay, the protease substrate is expressed in a solution that includes the elements of the ubiquitin proteasome pathway for degradation of the fragment. The assay measures the disappearance of the label attached to the fragment that results from cleavage by the protease.
1. A cell based assay to distinguish between two proteases, comprising:
providing a recombinant cell,
the recombinant cell having a vector that expresses a protease substrate having a label at its C-terminus, wherein said substrate is cleaved at a first site by a first protease generating a first C-terminus fragment and a first N-terminus fragment, and where said C-terminus fragment is degradable by the recombinant cell's ubiquitin proteasome pathway;
the protease substrate is also cleaved at a second site by a second protease generating a second C-terminus fragment and a second N-terminus fragment, and where said second C-terminus fragment is not degraded by the ubiquitin proteasome pathway;
contacting the recombinant cell expressing the protease substrate with the first protease and measuring the presence of the label;
contacting the recombinant cell expressing the protease substrate with the second protease and measuring the presence of the label;
comparing the difference in the presence of the label after contact with the first protease to the presence of the label after contact with the second protease.
2. The cell based assay of claim 1 , wherein the two proteases are BoNT/LC A and BoNT/LC E and the protease substrate is SNAP-25.
3. The cell based assay of claim 1 , further comprising the step of contacting a sample having one or more cells with a sample containing an unidentified protease; identifying the unidentified protease by measuring the degradation of the label in the cells.
4. The cell based assay of claim 1 , wherein the contacting step comprises exposing the recombinant cell to said first or second protease in a sample or inducing the production of said first or second protease in the cell from a vector.