Cellular libraries of peptide sequences (CLiPS) and methods of using the same
View Patent ↗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.
1. 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;
C 1 comprises a member of a library of candidate peptide substrates for the enzyme; and
D 1 and D 2 are first and second detectable moieties, wherein D 1 and D 2 are not the same and wherein when D 1 provides a detectable D 1 signal D 2 does not provide a detectable D 2 signal above a background level of a detectable D 2 signal; and
wherein D 1 -C 1 and D 2 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 D 1 signal; and detecting the presence or absence of a D 2 signal above a background level of a detectable D 2 signal,
wherein the presence of a detectable D 2 signal above a background level of a detectable D 2 signal identifies the member of the library of candidate peptide substrates as a peptide substrate for the enzyme.
2. The method of claim 1 , wherein said detecting is by fluorescence activated cell sorting.
3. The method of claim 1 , wherein an increase in the detectable D 2 signal relative to the detectable D 1 signal identifies the member of the library of candidate peptide substrates as a peptide substrate for the enzyme.
4. The method of claim 1 , wherein C 1 is [A-C s ] wherein A is an allosteric regulator for the enzyme and C s is a member of a library of candidate peptide substrates for the enzyme, and
wherein the presence of a detectable D 2 signal above a background level of a detectable D 2 signal identifies the member of the library of candidate peptide substrates as a peptide substrate for the enzyme.
5. 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;
C i is a member of a library of candidate inhibitor peptides; and
D 1 and D 2 are first and second detectable moieties, wherein D 1 and D 2 are different, and wherein prior to contacting the cell with the enzyme, the cell exhibits a detectable D 1 signal and does not exhibit a detectable D 2 signal above a background level of a detectable D 2 signal; and wherein D 1 -S-C i and D 2 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 D 1 signal and the presence or absence of a D 2 signal above a background level of a detectable D 2 signal,
wherein maintenance of the D 1 signal relative to the D 2 signal identifies C i as an inhibitor for the enzyme.
6. The method of claim 5 , wherein the cell is a member of a cell library enriched for expression of the peptide display scaffold by fluorescence activated cell sorting of cells exhibiting the D 1 signal.
7. The method of claim 5 , wherein said detecting the presence or absence of the D 1 signal and the D 2 signal is by fluorescence activated cell sorting.
8. The method of claim 5 , wherein the peptide display scaffold further comprising a linker between C i and TM.
9. The method of claim 5 , wherein D 1 and D 2 are affinity ligands.
10. The method of claim 1 , wherein the fusion protein further comprises at least one linker, wherein the linker is between C 1 and TM or D 2 and TM.
11. The method of claim 1 , wherein detectable moieties D 1 and D 2 are affinity tags.
12. The method of claim 1 , wherein D 1 -C 1 is located at an N-terminal domain of said fusion protein and D 2 is located at a C-terminal domain of said fusion protein.
13. The method of claim 1 , wherein D 1 -C 1 is located at a C-terminal domain of said fusion protein and D 2 is located at an N-terminal domain of said fusion protein.