Method for the detection of proteolytic enzymes

Abstract

The present invention provides an improved protease assay in which the proteases are detected on basis of their capability to cleave a modified pro-caspase which will yield an activated caspase which can then further be detected. Also part of the invention are the modified pro-caspases and kits comprising said modified pro-caspases.

1 . A modified pro-caspase derived from a pro-caspase by providing it with a recognition site which is cleavable by a protease different from the one which cleaves and activates the unmodified pro-caspase. 
     
     
         2 . The modified pro caspase according to  claim 1 , wherein said recognition site is an activation site cleavable by a protease different from the one which cleaves and activates the unmodified pro-caspase, said activation site replacing the original activation site of the pro-caspase. 
     
     
         3 . The modified pro-caspase according to  claim 2 , which is derived from pro-caspase-3 or pro-caspase-7. 
     
     
         4 . A kit for determining a protease, or its precursor after activation, in a sample, comprising a modified pro-caspase according to any of  claims 1 - 3  or  5 - 7 . 
     
     
         5 . The modified pro-caspase according to  claim 1 , wherein said protease is selected from the group consisting of serine proteases, cysteine proteases, aspartyl proteases and metalloproteases. 
     
     
         6 . The modified pro-caspase according to  claim 5 , wherein said protease is selected from the group consisting of aggrecanase, a disintegrin and metalloproteinase (ADAM) TS1, ADAM TS4, tumor necrosis factor alpha converting enzyme (TACE), β-site of APP cleaving enzyme (BACE), BACE 1, BACE 2, HIV protease and hepatitis C protease. 
     
     
         7 . The modified pro-caspase of  claim 3 , wherein said modification in pro-caspase 3 or pro-caspase 7 is a replacement of D175 in wild-type pro-caspase 3 or D198 in wild-type pro-caspase 7 by a sequence selected from the group of sequence GSDMELPLPRNITEGÊARGSVILTVKPIFEEF (SEQ ID NO: 1), GSPLAQÂVRSSSRSG (SEQ ID NO: 2) and GSKTEEISEVNL̂DAEFRHDS (SEQ ID NO: 3) wherein the ̂ symbol indicates the cleavage site in the physiological target.