GFP-SNAP25 Fluorescence Release Assay for Botulinum Toxin Protease Activity

Abstract

The present invention provides a nucleic acid molecule which contains a nucleotide sequence encoding a SNAP-25 substrate which includes (i) a green fluorescent protein; (ii) a first partner of an affinity couple; and (iii) a portion of SNAP-25 that includes a BoNT/A, BoNT/C1 or BoNT/E recognition sequence containing a cleavage site, where the cleavage site intervenes between the green fluorescent protein and the first partner of the affinity couple. Further provided herein is a nucleic acid molecule which contains a nucleotide sequence encoding a tagged toxin substrate which includes (i) a fluorescent protein; (ii) a first partner of an affinity couple; and (iii) a clostridial toxin recognition sequence containing a cleavage site, where the cleavage site intervenes between the fluorescent protein and the first partner of the affinity couple.

1 . A method of determining Botulinum toxin serotype A protease activity, comprising the steps of:
 (a) treating with a sample, in solution phase under conditions suitable for Botulinum toxin serotype A protease activity, a tagged toxin substrate comprising
 (i) a fluorescent protein; 
 (ii) a first partner of an affinity couple; and 
 (iii) a Botulinum toxin serotype A recognition sequence comprising a cleavage site, where the cleavage site intervenes between said fluorescent protein and said first partner of the affinity couple, such that a fluorescent cleavage product is generated when Botulinum toxin serotype A is present in said sample; 
   (b) contacting said treated sample with a second partner of the affinity couple, thereby forming stable complexes comprising said first and second partners of said affinity couple; and   (c) assaying the presence or amount of said fluorescent cleavage product in said treated sample, thereby determining Botulinum toxin serotype A protease activity.   
     
     
         2 . The method of  claim 1 , wherein said fluorescent protein is selected from the group green fluorescent protein (GFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and red fluorescent protein (RFP). 
     
     
         3 . The method of  claim 2 , wherein said fluorescent protein is GFP. 
     
     
         4 . The method of  claim 1 ,  2  or  3 , wherein said first partner of the affinity couple is selected from the group histidine tag, glutathione-S-transferase, maltose-binding protein, a biotinylation sequence, streptavidin, S peptide, S protein, FLAG, hemagluttinin (HA), c myc and AU1. 
     
     
         5 . The method of  claim 1 , wherein said first partner of the affinity couple is a histidine tag. 
     
     
         6 . The method of  claim 1 , wherein said Botulinum toxin serotype A recognition sequence comprises at least six consecutive residues of SNAP-25, said six consecutive residues comprising Gln-Arg, or a peptidomimetic thereof. 
     
     
         7 . The method of  claim 6 , wherein said Botulinum toxin serotype A recognition sequence comprises SEQ ID NO: 1, or a peptidomimetic thereof. 
     
     
         8 . The method of  claim 6 , wherein said Botulinum toxin serotype A recognition sequence comprises SEQ ID NO: 27, or a peptidomimetic thereof. 
     
     
         9 . The method of  claim 6 , wherein said Botulinum toxin serotype A recognition sequence comprises SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or a peptidomimetic thereof. 
     
     
         10 . The method of  claim 6 , wherein said Botulinum toxin serotype A recognition sequence comprises residues 134 to 206 of SEQ ID NO: 2, residues 137 to 206 of SEQ ID NO: 2, or a peptidomimetic thereof. 
     
     
         11 . The method of  claim 1 , wherein said substrate is cleaved with an activity of at least 1 nanomole/minute/milligram toxin. 
     
     
         12 . The method of  claim 1 , wherein said substrate is cleaved with an activity of at least 100 nanomoles/minute/milligram toxin. 
     
     
         13 . The method of  claim 1 , wherein said substrate is cleaved with an activity of at least 1000 nanomoles/minute/milligram toxin. 
     
     
         14 . The method of  claim 1 , wherein said second partner of the affinity couple is immobilized. 
     
     
         15 . The method of  claim 1 , wherein said second partner of the affinity couple comprises cobalt (Co2+). 
     
     
         16 . The method of  claim 1 , wherein said second partner of the affinity couple comprises nickel (Ni2+). 
     
     
         17 . The method of  claim 1 , further comprising separating said fluorescent cleavage product from said stable complexes prior to step (c). 
     
     
         18 . The method of  claim 17 , wherein said separating comprises applying said treated sample to a column, wherein said second partner of the affinity couple is immobilized on said column. 
     
     
         19 . The method of  claim 17 , wherein said separating comprises applying said treated sample to a filter plate, wherein said second partner of the affinity couple is immobilized on said filter plate. 
     
     
         20 . The method of  claim 1 , further comprising step (d) assaying the amount of uncleaved tagged toxin substrate in said treated sample. 
     
     
         21 . The method of  claim 1 , wherein said sample is isolated Botulinum toxin serotype A. 
     
     
         22 . The method of  claim 1 , wherein said sample is isolated Botulinum toxin serotype A light chain. 
     
     
         23 . The method of  claim 1 , wherein said formulated product is a formulated Botulinum toxin serotype A product. 
     
     
         24 . The method of  claim 1 , wherein said sample is a whole or partially purified cellular extract containing recombinantly expressed Botulinum toxin serotype A.