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US 2012/0058499 A1Published application
Bioluminescent Detection of Protease Activity
OwnerSteven J. Orcutt, Yan Yilin +3inventors
InventorsSteven J. Orcutt, Yan Yilin, Jian Wu, James Strickler, Tauseef R. Butt
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Abstract
Methods for bioluminescent detection of the activity of proteolytic enzymes including ubiquitin (Ub) and ubiquitin-like (Ubl) proteolytic enzymes are disclosed.
Claims18
A method for detecting the activity of a protease in a sample comprising
- (A) contacting said sample with
- (i) a protease substrate comprising
- (a) a first moiety comprising at least one ubiquitin (Ub) or a ubiquitin-like protein (Ubl), said first moiety comprising at its C-terminus a cleavage site for said protease, and
- (b) a second moiety comprising a luciferase substrate,
- wherein said first moiety is covalently linked at its C-terminus to said second moiety via an amide linkage;
- and
- (ii) luciferase,
- wherein said protease cleaves said protease substrate at the C-terminal end of the first moiety, thereby generating a free luciferase substrate;
- and
- (B) detecting luminescence in said sample, wherein luminescence is indicative of the activity of said protease.
7 more elements
A protease substrate comprising
- (A) a first moiety comprising at least one ubiquitin (Ub) or a ubiquitin-like protein (Ubl), said first moiety comprising at its C-terminus a cleavage site for a protease;
- and
- (B) a second moiety comprising a luciferase substrate,
- wherein the first moiety is covalently linked at its C-terminus to the second moiety via an amide linkage.
1 more element
A method for identifying the cleavage site of a protease comprising
- (A) contacting said protease with
- (i) at least one protease substrate comprising
- (a) a first moiety comprising at least one ubiquitin (Ub) or a ubiquitin-like protein (Ubl), said first moiety comprising at its C-terminus an amino acid sequence of interest, and
- (b) a second moiety comprising a luciferase substrate,
- wherein said first moiety is covalently linked at its C-terminus to said second moiety via an amide linkage;
- and
- (ii) luciferase,
- wherein said protease cleaves said protease substrate at the C-terminal end of the first moiety, thereby generating a free luciferase substrate;
- and
- (B) detecting luminescence in said sample, wherein the presence of luminescence is indicative that the amino acid sequence of interest comprises said cleavage site.
7 more elements
Dependencies are inferred from the claim text.