Enzyme activation protease assay
AssigneeDiscoveRx Corporation
InventorsPyare Khanna, Peter Fung, Joseph L. Horecka
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Abstract
A protein reagent is provided for measuring protease enzyme activity in a sample. The protein reagent comprises two inhibition entities joined by a linker comprised of an indicator enzyme donor and an amino acid sequence susceptible to enzymatic cleavage. The protein reagent is substantially inhibited from binding to the cognate enzyme acceptor fragment, while the product of the enzymatic cleavage binds to the cognate enzyme acceptor fragment to form a functional indicator enzyme. The indicator enzyme activity is related to the protease enzyme activity of the sample.
Claims20
A method for determining target protease activity in a sample, employing a protein reagent comprising first and second hindering entities, the hindering entities linked together by an enzyme donor (“ED”) fragment and an amino acid sequence comprising a covalent bond susceptible to cleavage by said target protease activity, wherein said ED in the absence of at least one hindering entity is able to form an active indicator enzyme with an enzyme acceptor (“EA”) fragment, wherein cleavage of the cleavable sequence by the target protease produces a protein reagent fragment active with said EA to produce an active indicator enzyme, wherein indicator enzyme activity is related to said target protease activity, and wherein said protein reagent fragment is at least 5 times as active as said protein reagent in complexing with said EA in forming active indicator enzyme, said method comprising:
- combining said protein reagent, said sample, enzyme acceptor and indicator enzyme substrate for a time sufficient for said target protease activity to cleave said covalent bond;
- and
- measuring the indicator enzyme activity as an indication of the protease activity.
A method for determining protease activity in a sample, employing a protein reagent comprising first and second hindering entities, the hindering entities linked together by an enzyme donor (“ED”) fragment of β-galactosidase and an amino acid sequence comprising a covalent bond specifically susceptible to cleavage by said protease activity, wherein said ED in the absence of at least one hindering entity is able to form with an enzyme acceptor fragment of β-galactosidase to form an active β-galactosidase, wherein cleavage of the cleavable amino acid seciuence by said target protease produces a protein reagent fragment active with said enzyme acceptor to produce an active β-galactosidase, wherein β-galactosidase activity is related to said protease activity, and wherein said protein reagent fragment is at least 5 times as active as said protein reagent in complexing with said EA in forming active β-galactosidase, said method comprising:
- combining said protein reagent, said sample, enzyme acceptor and β-gaiactosidase substrate for a time sufficient for said enzyme activity to cleave said amino acid sequence;
- and
- measuring the β-galactosidase activity as an indication of the enzyme activity.
A method for determining target protease activity in a sample, employing a protein reagent comprising first and second hindering entities, the hindering entities linked together by an enzyme donor (“ED”) fragment of β-galactosidase and an amino acid sequence comprising a covalent bond susceptible to cleavage by said target protease activity, said protein reagent substantially inactive in complexing with an enzyme acceptor fragment of β-galactosidase to form an active β-galactosidase, wherein cleavage of the cleavable amino acid seciuence by said target protease produces a protein reagent fragment comprising said ED in the absence of at least one hindering entity to form an active β-galactosidase with an enzyme acceptor fragment and said protein reagent fragment is of at least about 125 kDa active with said enzyme acceptor, wherein indicator enzyme activity is related to said target protease activity, and wherein said hindering entities result in at least a 5-fold reduction in β-galactosidase activity in said sample, said method comprising:
- combining said protein reagent, said sample, enzyme acceptor and β-galactosidase substrate for a time sufficient for said target protease activity to cleave said covalent bond;
- and
- measuring the indicator enzyme activity as an indication of the protease activity.
Dependencies are inferred from the claim text.