Cellular libraries of peptide sequences (CLiPS) and methods of using the same
AssigneeThe Regents of the University of California
InventorsPatrick Sean Daugherty, Kevin T. Boulware
Life of this patent
Estimating the term…
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.
Claims13
A method for identifying a peptide substrate for an enzyme, comprising:
- contacting a cell with an enzyme, the cell comprising a cell outer membrane and expressing a peptide display scaffold, the peptide display scaffold comprising
- a fusion protein comprising the formula:
- wherein
- TM is a circularly permuted bacterial outer membrane protein;
- C1 comprises a member of a library of candidate peptide substrates for the enzyme;
- and
- D1 and D2 are first and second detectable moieties, wherein D1 and D2 are not the same and wherein when D1 provides a detectable D1 signal D2 does not provide a detectable D2 signal above a background level of a detectable D2 signal;
- and
- wherein D1-C1 and D2 are accessible at the extracellular surface of the cell outer membrane and the N- or C-terminus of the fusion protein is accessible at the extracellular surface of the cell outer membrane, wherein prior to contacting the cell with the enzyme, the cell exhibits a detectable D1 signal;
- and detecting the presence or absence of a D2 signal above a background level of a detectable D2 signal,
- wherein the presence of a detectable D2 signal above a background level of a detectable D2 signal identifies the member of the library of candidate peptide substrates as a peptide substrate for the enzyme.
A method for identifying a peptide inhibitor for an enzyme, comprising:
- contacting a cell expressing a peptide display scaffold with an enzyme, wherein the peptide display scaffold comprises a fusion protein comprising the formula:
- wherein
- TM is a circularly permuted bacterial outer membrane protein;
- S is a substrate for the enzyme;
- Ci is a member of a library of candidate inhibitor peptides;
- and
- D1 and D2 are first and second detectable moieties, wherein D1 and D2 are different, and wherein prior to contacting the cell with the enzyme, the cell exhibits a detectable D1 signal and does not exhibit a detectable D2 signal above a background level of a detectable D2 signal;
- and wherein D1-S-Ci and D2 are accessible at the extracellular surface of the cell outer membrane and the N- or C-terminus of the fusion protein is accessible at the extracellular surface of the cell outer membrane;
- and
- detecting the presence or absence of a D1 signal and the presence or absence of a D2 signal above a background level of a detectable D2 signal,
- wherein maintenance of the D1 signal relative to the D2 signal identifies Ci as an inhibitor for the enzyme.
Dependencies are inferred from the claim text.