Cellular Libraries of Peptide Sequences (CLiPS) and Methods of Using the Same
AssigneeThe Regents of the University of California
InventorsPatrick Sean Daugherty, Kevin Todd Boulware
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Abstract
The present invention provides compositions including peptide display scaffolds that present at least one candidate peptide and at least one detectable moiety in at least one of the N-terminal and C-terminal candidate peptide presenting domains that when expressed in a cell are accessible at a surface of the cell outermembrane. In addition, the present invention also provides kits and methods for screening a library of cells presenting the candidate peptides in peptide display scaffolds to identify a ligand for an enzyme.
Claims23
1-46. (canceled)
A method of identifying a substrate for a target protease, the method comprising:
- contacting a plurality of cells with a background mixture,
- wherein the background mixture does not include the target protease and wherein each of the plurality of cells expresses a peptide display scaffold;
- and
- enriching the plurality of cells for cells that are not cleaved by background mixture,
- wherein the peptide display scaffold comprises a fusion protein having the structure [D1-C1]-TM-[D2],
- wherein TM is a circularly permuted transmembrane protein, wherein C1 comprises a candidate substrate of the target protease, wherein D1 is a first detectable moiety and is a heterologous peptide or polypeptide, wherein D2 is a second detectable moiety and is a heterologous peptide or polypeptide, wherein D1 and D2 are not the same;
- and wherein D1, D2, and C1 are exposed at an extracellular surface of the cell outer membrane.
A method of identifying a substrate for a target protease, the method comprising:
- contacting a plurality of cells with a background mixture, wherein the background mixture does not include the target protease and wherein each of the plurality of cells expresses a peptide display scaffold;
- enriching the plurality of cells that are not cleaved by the background mixture;
- contacting the enriched plurality of cells with the target protease;
- isolating the cells from the enriched plurality of cells that are cleaved by the target protease;
- and
- amplifying the isolated cells,
- wherein the peptide display scaffold comprises a fusion protein having the structure [D1-C1] TM-[D2],
- wherein TM is a circularly permuted transmembrane protein, wherein C1 comprises a candidate substrate for the target protease, wherein D1 is a first detectable moiety and is a heterologous peptide or polypeptide, wherein D2 is a second detectable moiety and is a heterologous peptide or polypeptide, wherein D1 and D2 are not the same;
- and wherein D1, D2, and C1 are exposed at an extracellular surface of the cell outer membrane.
Dependencies are inferred from the claim text.