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

• US 9,234,847 B2
• Filed: 12/23/2009
• Issued: 01/12/2016
• 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): Vivlemore, Tracy
• Assistant Examiner(s): Weiler, Karen S
• Application Number:
  US12/646,323
• Publication Number:
  US 20100173349A1
• Time in Patent Office: 2,211 Days
• Field of Search: None
• US Class Current: 1/1
• 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:

a D₁ moiety positioned N-terminal to a C₁ moiety in the fusion protein, which C₁ moiety is positioned N-terminal to a TM moiety in the fusion protein, which TM moiety is positioned N-terminal to a D₂ moiety in the fusion protein;

or

a D₂ moiety positioned N-terminal to a TM moiety in the fusion protein, which TM moiety is positioned N-terminal to a C₁ moiety in the fusion protein, which C₁ moiety is positioned N-terminal to a D₁ moiety in the fusion protein;

wherein

the TM moiety is a circularly permuted bacterial outer membrane protein;

the C₁ moiety comprises a candidate peptide; and

the D₁ and D₂ moieties are first and second detectable moieties, wherein the D₁ and D₂ moieties are not the same, wherein the D₁ moiety and the D₂ 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 D₁, C₁ and D₂ 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 D₂ moiety and the TM moiety.

3. The peptide display scaffold of claim 1, wherein detectable moieties D₁ and D₂ 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 C₂ moiety between the TM moiety and the D₂ moiety, wherein the fusion protein comprises

the D₁ moiety positioned N-terminal to the C₁ moiety in the fusion protein, which C₁ moiety is positioned N-terminal to the TM moiety in the fusion protein, which TM moiety is positioned N-terminal to the C₂ moiety in the fusion protein, which C₂ moiety is positioned N-terminal to the D₂ moiety in the fusion protein;

or

the D₂ moiety positioned N-terminal to the C₂ moiety in the fusion protein, which C₂ moiety is positioned N-terminal to the TM moiety in the fusion protein, which TM moiety is positioned N-terminal to the C₁ moiety in the fusion protein, which C₁ moiety is positioned N-terminal to the D₁ moiety in the fusion protein;

wherein the C₂ moiety comprises a candidate peptide, and the C₁ and C₂ moieties are not the same, and wherein when the fusion protein is expressed in the cell, the D₁, C₁, C₂ and D₂ 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 C₁ moiety and the TM moiety or between the C₂ moiety and the TM moiety.

6. The peptide display scaffold of claim 4, wherein detectable moieties D₁ and D₂ 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 D₁ signal and a D₂ signal, wherein said detecting indicates whether C₂ comprises a peptide ligand for the enzyme.

8. The peptide display scaffold of claim 1, wherein the C₁ 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 C₁ moiety and the TM moiety and a linker between the D₂ moiety and the TM moiety.

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

11. 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.

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

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

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

15. The peptide display scaffold of claim 4, wherein the C₁ 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 C₁ moiety and the TM moiety and a linker between the C₂ moiety and the TM moiety.

17. The peptide display scaffold of claim 4, wherein at least one of detectable moieties D₁ and D₂ is an affinity tag.

18. The peptide display scaffold of claim 4, 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.

19. The peptide display scaffold of claim 4, wherein at least one of detectable moieties D₁ and D₂ is a fluorescent protein.

20. The peptide display scaffold of claim 4, wherein the D₁ and C_i moieties are located at an N-terminal domain of the fusion protein and the C₂ and D₂ moieties are located at a C-terminal domain of the fusion protein.

21. The peptide display scaffold of claim 4, wherein the D₁ and C_i moieties are located at a C-terminal domain of the fusion protein and the C₂ and D₂ 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 D₁ signal and a D₂ signal, wherein said detecting indicates whether C₁ comprises a peptide ligand for the enzyme.