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 peptide display scaffold, comprising a fusion protein comprising:
a D 1 moiety positioned N-terminal to a C 1 moiety in the fusion protein, which C 1 moiety is positioned N-terminal to a TM moiety in the fusion protein, which TM moiety is positioned N-terminal to a D 2 moiety in the fusion protein; or
a D 2 moiety positioned N-terminal to a TM moiety in the fusion protein, which TM moiety is positioned N-terminal to a C 1 moiety in the fusion protein, which C 1 moiety is positioned N-terminal to a D 1 moiety in the fusion protein; wherein
the TM moiety is a circularly permuted bacterial outer membrane protein;
the C 1 moiety comprises a candidate peptide; and
the D 1 and D 2 moieties are first and second detectable moieties, wherein the D 1 and D 2 moieties are not the same, wherein the D 1 moiety and the D 2 moiety are each a heterologous peptide or polypeptide relative to the circularly permuted bacterial outer membrane protein, and wherein, when the fusion protein is expressed in a cell comprising a cell outer membrane, the D 1 , C 1 and D 2 moieties are exposed at an extracellular surface of the cell outer membrane and one or both of the N- and C-termini of the fusion protein are exposed at the extracellular surface of the cell outer membrane.
2. The peptide display scaffold of claim 1 , wherein the fusion protein comprises at least one linker, wherein the linker is between the C i moiety and the TM moiety or between the D 2 moiety and the TM moiety.
3. The peptide display scaffold of claim 1 , wherein detectable moieties D 1 and D 2 are first and second affinity tags which are not the same.
4. The peptide display scaffold of claim 1 , wherein the fusion protein comprises a C 2 moiety between the TM moiety and the D 2 moiety, wherein the fusion protein comprises
the D 1 moiety positioned N-terminal to the C 1 moiety in the fusion protein, which C 1 moiety is positioned N-terminal to the TM moiety in the fusion protein, which TM moiety is positioned N-terminal to the C 2 moiety in the fusion protein, which C 2 moiety is positioned N-terminal to the D 2 moiety in the fusion protein; or
the D 2 moiety positioned N-terminal to the C 2 moiety in the fusion protein, which C 2 moiety is positioned N-terminal to the TM moiety in the fusion protein, which TM moiety is positioned N-terminal to the C 1 moiety in the fusion protein, which C 1 moiety is positioned N-terminal to the D 1 moiety in the fusion protein;
wherein the C 2 moiety comprises a candidate peptide, and the C 1 and C 2 moieties are not the same, and wherein when the fusion protein is expressed in the cell, the D 1 , C 1 , C 2 and D 2 moieties are exposed at the extracellular surface of the cell outer membrane.
5. The peptide display scaffold of claim 4 , wherein the fusion protein comprises at least one linker, wherein the linker is between the C 1 moiety and the TM moiety or between the C 2 moiety and the TM moiety.
6. The peptide display scaffold of claim 4 , wherein detectable moieties D 1 and D 2 are first and second affinity tags which are not the same.
7. A method for identifying a peptide ligand of an enzyme, comprising:
contacting a cell expressing a peptide display scaffold according to claim 4 with an enzyme; and
detecting the presence or absence of a D 1 signal and a D 2 signal, wherein said detecting indicates whether C 2 comprises a peptide ligand for the enzyme.
8. The peptide display scaffold of claim 1 , wherein the C 1 moiety comprises a member of a library of candidate peptide substrates for an enzyme.
9. The peptide display scaffold of claim 1 , wherein the fusion protein comprises a linker between the C 1 moiety and the TM moiety and a linker between the D 2 moiety and the TM moiety.
10. The peptide display scaffold of claim 1 , wherein at least one of detectable moieties D 1 and D 2 is an affinity tag.
11. The peptide display scaffold of claim 1 , wherein at least one of detectable moieties D 1 and D 2 is a binding peptide selected from the group consisting of a streptavidin binding peptide and a monocytic adaptor protein (MONA) binding peptide.
12. The peptide display scaffold of claim 1 , wherein at least one of detectable moieties D 1 and D 2 is a fluorescent protein.
13. The peptide display scaffold of claim 1 , wherein the D 1 and C 1 moieties are located at an N-terminal domain of the fusion protein and the D 2 moiety is located at a C-terminal domain of the fusion protein.
14. The peptide display scaffold of claim 1 , wherein the D 1 and C 1 moieties are located at a C-terminal domain of the fusion protein and the D 2 moiety is located at an N-terminal domain of the fusion protein.
15. The peptide display scaffold of claim 4 , wherein the C 1 moiety comprises a member of a library of candidate peptide substrates for an enzyme.
16. The peptide display scaffold of claim 4 , wherein the fusion protein comprises a linker between the C 1 moiety and the TM moiety and a linker between the C 2 moiety and the TM moiety.
17. The peptide display scaffold of claim 4 , wherein at least one of detectable moieties D 1 and D 2 is an affinity tag.
18. The peptide display scaffold of claim 4 , wherein at least one of detectable moieties D 1 and D 2 is a binding peptide selected from the group consisting of a streptavidin binding peptide and a monocytic adaptor protein (MONA) binding peptide.
19. The peptide display scaffold of claim 4 , wherein at least one of detectable moieties D 1 and D 2 is a fluorescent protein.
20. The peptide display scaffold of claim 4 , wherein the D 1 and C i moieties are located at an N-terminal domain of the fusion protein and the C 2 and D 2 moieties are located at a C-terminal domain of the fusion protein.
21. The peptide display scaffold of claim 4 , wherein the D 1 and C i moieties are located at a C-terminal domain of the fusion protein and the C 2 and D 2 moieties are located at an N-terminal domain of the fusion protein.
22. The peptide display scaffold of claim 1 , wherein the circularly permuted bacterial outer membrane protein is a circularly permuted OmpX.
23. The peptide display scaffold of claim 4 , wherein the circularly permuted bacterial outer membrane protein is a circularly permuted OmpX.
24. The method of claim 7 , wherein the circularly permuted bacterial outer membrane protein is a circularly permuted OmpX.
25. A method for identifying a peptide ligand of an enzyme, comprising:
contacting a cell expressing a peptide display scaffold according to claim 1 with an enzyme; and
detecting the presence or absence of a D 1 signal and a D 2 signal, wherein said detecting indicates whether C 1 comprises a peptide ligand for the enzyme.