Cellular libraries of peptide sequences (CLiPS) and methods of using the same

• US 10,017,757 B2
• Filed: 07/21/2015
• Issued: 07/10/2018
• Est. Priority Date: 08/31/2005
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Daugherty, Patrick Sean, Boulware, Kevin Todd
• Primary Examiner(s): Boesen, Christian C
• Application Number:
  US14/805,284
• Publication Number:
  US 20160010084A1
• Time in Patent Office: 1,085 Days
• Field of Search: None
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C07K 2319/03 containing a transmembrane segment
• CPC Class Codes: C07K 2319/22 containing a Strep-tag
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1044 Preparation or screening of libraries displayed on scaffold proteins
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence

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.

Claims

1. A peptide display scaffold comprising a fusion protein comprising the formula:

[D₁-C₁]-TM-[D₂],

wherein TM is a circularly permuted transmembrane protein;

wherein C₁ comprises a candidate peptide;

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, when the peptide display scaffold is expressed in a host cell, are exposed at an extracellular surface of the host cell outer membrane,

wherein the circularly permuted transmembrane protein is a circularly permuted OmpX, OmpA, or OmpT.

2. The peptide display scaffold of claim 1, wherein the fusion protein comprises at least one linker, wherein the linker is between C₁ and TM or between D₂ and TM.

3. The peptide display scaffold of claim 1, wherein detectable moieties D₁ and D₂ are affinity tags.

4. The peptide display scaffold of claim 1, 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.

5. The peptide display scaffold of claim 1, wherein the C₁ comprises a member of a library of candidate peptide substrates for an enzyme.

6. The peptide display scaffold of claim 1, wherein the fusion protein comprises a linker between the C₁ and the TM and a linker between the D₂ and the TM.

7. The peptide display scaffold of claim 1, wherein at least one of detectable moieties D₁ and D₂ is an affinity tag.

8. The peptide display scaffold of claim 1, wherein at least one of detectable moieties D₁ and D₂ is a binding peptide selected from the group consisting of a streptavidin binding peptide and a monocytic adaptor protein (MONA) binding peptide.

9. The peptide display scaffold of claim 1, wherein at least one of detectable moieties D₁ and D₂ is a fluorescent protein.

10. The peptide display scaffold of claim 1, wherein the D₁ and C₁ are located at an N-terminal domain of the fusion protein and the D₂ is located at a C-terminal domain of the fusion protein.

11. The peptide display scaffold of claim 1, wherein the D₁ and C₁ are located at a C-terminal domain of the fusion protein and the D₂ is located at an N-terminal domain of the fusion protein.

12. The peptide display scaffold of claim 1, wherein the circularly permuted transmembrane protein is a circularly permuted bacterial outer membrane protein.

13. The peptide display scaffold of claim 1, wherein the fusion protein comprises a C₂ between TM and D₂, wherein the fusion protein comprises the formula:

[D₁-C₁]-TM-[C₂-D₂],

wherein C₂ comprises a candidate peptide, and C₁ and C₂ are not the same; and

wherein D₁, D₂, C₁, and C₂, when the peptide display scaffold is expressed in a host cell, are exposed at an extracellular surface of the host cell outer membrane.

14. The peptide display scaffold of claim 13, wherein the fusion protein comprises at least one linker, wherein the linker is between C₁ and TM or between C₂ and TM.

15. The peptide display scaffold of claim 13, wherein detectable moieties D₁ and D₂ are affinity tags.

16. The peptide display scaffold of claim 13, 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.

17. The peptide display scaffold of claim 13, wherein the C₁ comprises a member of a library of candidate peptide substrates for an enzyme.

18. The peptide display scaffold of claim 13, wherein the fusion protein comprises a linker between the C₁ and the TM and a linker between the C₂ and the TM.

19. The peptide display scaffold of claim 13, wherein at least one of detectable moieties D₁ and D₂ is an affinity tag.

20. The peptide display scaffold of claim 13, wherein at least one of detectable moieties D₁ and D₂ is a binding peptide selected from the group consisting of a streptavidin binding peptide and a monocytic adaptor protein (MONA) binding peptide.

21. The peptide display scaffold of claim 13, wherein at least one of detectable moieties D₁ and D₂ is a fluorescent protein.

22. The peptide display scaffold of claim 13, wherein the D₁ and C₁ are located at an N-terminal domain of the fusion protein and the D₂ and C₂ are located at a C-terminal domain of the fusion protein.

23. The peptide display scaffold of claim 13, wherein the D₁ and C₁ are located at a C-terminal domain of the fusion protein and the D₂ and C₂ are located at an N-terminal domain of the fusion protein.

24. The peptide display scaffold of claim 13, wherein the circularly permuted transmembrane protein is a circularly permuted bacterial outer membrane protein.

25. The peptide display scaffold of claim 13, wherein the circularly permuted transmembrane protein is a circularly permuted OmpX.

26. A method of identifying an allosteric regulator for an enzyme, comprising:

contacting a cell expressing a peptide display scaffold according to claim 13 with an enzyme, wherein C₁ is a known substrate for the enzyme; and

detecting the presence or absence of a D₁ signal and a D₂ signal, wherein said detecting indicates whether C₂ comprises an allosteric regulator for the enzyme.

27. A method of identifying an inhibitor for an enzyme, comprising:

contacting a cell expressing a peptide display scaffold according to claim 13 with an enzyme, wherein C₁ is a known substrate for the enzyme; and

detecting the presence or absence of a D₁ signal and a D₂ signal, wherein said detecting indicates whether C₂ comprises an inhibitor for the enzyme.

28. A method of identifying a substrate for 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 D₁ signal and a D₂ signal, wherein said detecting indicates whether C₁ comprises a substrate for the enzyme.

29. 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 D₁ signal and a D₂ signal, wherein said detecting indicates whether C₁ comprises a peptide ligand for the enzyme.