Protease EFC cell surface fusion protein assay
View Patent ↗Methods and reagents are provided for measuring protease activity. The reagent comprises a surface to which is linked an enzyme donor fragment through a protease recognition sequence, where the enzyme donor fragment complexes with an enzyme acceptor fragment to form an active indicator enzyme when the enzyme donor fragment is cleaved from said surface. Conveniently, the surface is a cell membrane surface where the reagent is expressed in the cell. The method comprises bringing together the reagent, the protease, the enzyme acceptor and substrate for the indicator enzyme and measuring the indicator enzyme activity as a measure of the protease activity. The method finds application in screening for compounds modulating the activity of the protease.
1. A method for determining target protease activity in a sample, employing a transfected cell comprising a hindered protein reagent, said hindered protein reagent comprising a membrane bound protein component bound to a cell membrane surface, said protein component linked to an enzyme donor fragment of β-galactosidase by a recognition sequence of from 4 to 12 amino acids susceptible to cleavage by said target protease activity to produce an enzyme donor fragment, said cell surface of said hindered protein reagent inhibiting the formation of a complex between said enzyme donor fragment of β-galactosidase and a partner enzyme acceptor fragment of β-galactosidase resulting in a low background signal, wherein cleavage produces a protein reagent fragment active with said enzyme acceptor fragment of said β-galactosidase to produce an active β-galactosidase enzyme resulting in an increase in β-galactosidase enzyme activity, wherein β-galactosidase enzyme activity is related to said target protease activity, said method comprising:
combining said transfected cell, said sample, enzyme acceptor fragment of β-galactosidase and β-galactosidase enzyme substrate for a time sufficient for said target protease activity to cleave said recognition sequence; and
measuring the β-galactosidase enzyme activity as an indication of the protease activity.
2. The method according to claim 1 , wherein said enzyme donor fragment and said enzyme acceptor fragment are free of complexing fragments and are fragments of said enzyme that independently complex to form an active β-galactosidase enzyme.
3. The method according to claim 1 , wherein said enzyme donor fragment and said enzyme acceptor fragment consist of fusion proteins, said enzyme donor fragment comprised of a first binding protein and a first fragment of said β-galactosidase enzyme and said enzyme acceptor fragment comprised of a second binding protein and a second fragment of said β-galactosidase enzyme, wherein said first and second fragments of said β-galactosidase enzyme do not independently complex to form an active enzyme and upon complex formation of said first and second binding proteins an active β-galactosidase enzyme is formed.
4. The method according to claim 3 , wherein first and second binding proteins require a supplemental reagent for binding.
5. The method according to claim 1 , wherein said protein reagent is anchored to said cell membrane by an amino acid sequence spanning said cell membrane.
6. The method according to claim 1 , wherein said enzyme donor is of from about 37 to 120 amino acids.
7. The method according to claim 1 , wherein said recognition sequence is within 50 amino acids of said enzyme donor.
8. A method for determining protease activity in a sample, employing a transfected cell comprising a hindered protein reagent, said hindered protein reagent comprising a membrane bound protein component bound to an intact cell membrane surface hindering entity, said protein component linked to an enzyme donor fragment of β-galactosidase by an amino acid recognition sequence of from 4 to 12 amino acids specifically susceptible to cleavage by said target protease activity to produce an enzyme donor fragment, said membrane cell surface hindering entity of said hindered protein reagent inhibiting the formation of a complex between said enzyme donor fragment of β-galactosidase and a partner enzyme acceptor fragment of β-galactosidase resulting in a low background signal, wherein cleavage produces a protein reagent fragment active with said enzyme acceptor fragment of β-galactosidase to produce an active β-galactosidase resulting in an increase in β-galactosidase activity, wherein β-galactosidase activity is related to said protease activity, said method comprising:
combining said transfected cell, said sample, enzyme acceptor fragment of said β-galactosidase and β-galactosidase substrate for a time sufficient for said protease activity to cleave said amino acid sequence; and
measuring the β-galactosidase activity as an indication of the protease activity.
9. The method according to claim 8 , wherein said amino acid recognition sequence is within 50 amino acids of said enzyme donor.
10. The method according to claim 8 , wherein said protease is a serine/threonine hydrolase.
11. The method according to claim 8 , wherein said protease is a metalloproteinase.