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 cell comprising a peptide display scaffold disposed in an outer membrane of the cell, the peptide display scaffold comprising a fusion protein comprising the formula:
[D 1 -C 1 ]-TM-[D 2 ]
wherein TM is a circularly permuted OmpA or OmpX protein;
C 1 comprises a candidate peptide; and
D 1 and D 2 are first and second detectable moieties, wherein D 1 is a heterologous peptide or polypeptide and D 2 is a heterologous peptide or polypeptide, wherein D 1 and D 2 are not the same and wherein D 1 -C 1 and D 2 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 cell of claim 1 , wherein the fusion protein comprises at least one linker, wherein the linker is between C i and TM or between D 2 and TM.
3. The cell of claim 1 , wherein the fusion protein comprises a linker between C 1 and TM and a linker between D 2 and TM.
4. The cell of claim 1 , wherein detectable moieties D 1 and D 2 are first and second affinity tags which are not the same.
5. The cell of claim 1 , wherein C 1 comprises a member of a library of candidate peptide substrates for an enzyme.
6. The cell of claim 1 , wherein at least one of detectable moieties D 1 and D 2 is a fluorescent protein.
7. The cell of claim 1 , wherein D 1 -C 1 is located at an N-terminal domain of the fusion protein and D 2 is located at a C-terminal domain of the fusion protein.
8. The cell of claim 1 , wherein C 1 -D 1 is located at a C-terminal domain of the fusion protein and D 2 is located at an N-terminal domain of the fusion protein.
9. The cell of claim 1 , wherein the fusion protein comprises a C 2 between TM and D 2 , wherein the fusion protein comprises the formula
[D 1 -C 1 ]-TM-[C 2 -D 2 ]
wherein C 2 comprises a candidate peptide, and C 1 and C 2 are not the same, and wherein D 1 -C 1 and C 2 -D 2 are exposed at the extracellular surface of the cell outer membrane.
10. The cell of claim 9 , wherein the fusion protein comprises at least one linker, wherein the linker is between C 1 and TM or between C 2 and TM.
11. The cell of claim 9 , wherein the fusion protein comprises a linker between C 1 and TM and a linker between C 2 and TM.
12. The cell of claim 9 , wherein detectable moieties D 1 and D 2 are first and second affinity tags which are not the same.
13. The cell of claim 9 , wherein C 1 comprises a member of a library of candidate peptide substrates for an enzyme.
14. The cell of claim 9 , wherein at least one of detectable moieties D 1 and D 2 is a fluorescent protein.
15. The cell of claim 9 , wherein D 1 -C 1 is located at an N-terminal domain of the fusion protein and C 2 -D 2 is located at a C-terminal domain of the fusion protein.
16. The cell of claim 9 , wherein C 1 -D 1 is located at a C-terminal domain of the fusion protein and D 2 -C 2 is located at an N-terminal domain of the fusion protein.
17. A method for identifying a peptide substrate for an enzyme, the method comprising:
contacting a cell according to claim 1 with an enzyme; and
determining whether there is an increase in a D 2 signal from detectable moiety D 2 relative to a D 1 signal from detectable moiety D 1 , wherein an increase in the D 2 signal relative to the D 1 signal indicates that C 1 comprises a substrate for the enzyme.
18. A method for identifying an inhibitor for an enzyme, comprising:
contacting a cell according to claim 9 with an enzyme, wherein C 1 comprises a substrate for the enzyme and C 2 comprises a candidate inhibitor for the enzyme; and
detecting the presence or absence of a D 1 signal and a D 2 signal, wherein when C 1 comprises a substrate for the enzyme and C 2 comprises a candidate inhibitor for the enzyme, maintenance of the D 1 signal relative to the D 2 signal following contact with the enzyme indicates that C 2 comprises an inhibitor for the enzyme.
19. A method for identifying a peptide substrate for an enzyme, comprising:
contacting a cell according to claim 9 with an enzyme, wherein C 1 comprises an allosteric regulator for the enzyme and C 2 comprises a candidate substrate for the enzyme; and
determining whether there is a change in a D 2 signal from detectable moiety D 2 relative to a D 1 signal from detectable moiety D 1 , wherein a change in the D 2 signal relative to the D 1 signal indicates that C 2 comprises a substrate for the enzyme.
20. A method for identifying an allosteric regulator for an enzyme, comprising:
contacting a cell according to claim 9 with an enzyme, wherein C 1 comprises a known substrate for the enzyme and C 2 comprises a candidate allosteric regulator for the enzyme; and
determining whether there is a change in a D 2 signal from detectable moiety D 2 relative to a D 1 signal from detectable moiety D 1 , wherein a change in the D 2 signal relative to the D 1 signal indicates that C 2 comprises an allosteric regulator for the enzyme.