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.
Claims31
A method for determining target protease activity in a sample, employing a protein reagent comprising first and second hindering entities linked together by an enzyme donor fragment and a covalent bond susceptible to cleavage by said target protease activity, said protein reagent substantially inactive in complexing with an enzyme acceptor fragment to form an active indicator enzyme, wherein cleavage produces a protein reagent fragment active with said enzyme acceptor to produce an active indicator enzyme, wherein indicator enzyme activity is related to said target protease activity, 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 linked together by an enzyme donor fragment of β-galactosidase and an amino acid sequence specifically susceptible to cleavage by said 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 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, said method comprising:
- combining said protein reagent, said sample, enzyme acceptor and β-galactosidase 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 linked together by an enzyme donor fragment of β-galactosidase and 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 produces a protein reagent fragment of at least about 125 kDa active with said enzyme acceptor to produce an active indicator enzyme, wherein indicator enzyme activity is related to said target protease activity, 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.
A water soluble protein reagent consisting essentially of a protein of at least about 25 kDa and not more than about 200 kDa joined to a recognition sequence recognized by a protease for proteolytic cleavage joined directly or through a linker of not more than about 50 amino acids to an enzyme donor fragment of an indicator enzyme joined to a protein of at least about 25 kDa, wherein said enzyme donor fragment is substantially unable to complex with an enzyme acceptor fragment to form an active indicator enzyme as part of said protein reagent and is able to complex with an enzyme acceptor fragment to form an active indicator enzyme when said recognition sequence is cleaved.
A protein reagent of at least 10 kDa and not more than about 150 kDa consisting essentially of a linker of at least about 10 amino acids comprising a linking entity for linking to a surface or liposome joined to a recognition sequence recognized by a protease for proteolytic cleavage joined directly or through a linker of not more than about 50 amino acids to an enzyme donor fragment of an indicator enzyme joined to a protein of at least about 25 kDa, wherein said enzyme donor fragment is substantially unable to complex with an enzyme acceptor fragment to form an active indicator enzyme as part of said protein reagent linked to said surface or said liposome and is able to complex with an enzyme acceptor fragment to form an active indicator enzyme when said recognition sequence is cleaved.
A kit according to claim 26, wherein said gene construct is intracellular.
A reagent consisting essentially of a protein of at least about 25 kDa joined to an enzyme donor fragment joined directly or through a linker of not more than about 50 amino acids to a recognition sequence recognized by a protease for proteolytic cleavage, said recognition sequence joined to a surface or liposome by a linker of not more than about 150 amino acids, wherein said enzyme donor fragment is substantially unable to complex with an enzyme acceptor fragment to form an active enzyme as part of said protein reagent and is able to complex with an enzyme acceptor fragment to form an active enzyme when said recognition sequence is cleaved.
Dependencies are inferred from the claim text.