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

• US 20070065878A1
• Filed: 08/30/2006
• Published: 03/22/2007
• 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, Boulware, Kevin
• Granted Patent: US 7,666,817 B2
• Time in Patent Office: Days
• US Class Current: 435/7.100
• 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 method for screening a library of cells presenting candidate peptides in peptide display scaffolds to identify a peptide substrate for an enzyme, comprising:

contacting an enzyme with a cell library enriched for expression of peptide display scaffolds, wherein each peptide display scaffold comprises;

wherein

TM is a transmembrane protein;

C is a candidate peptide; and

D is a detectable moiety, wherein the cells of the cell library exhibit

a D signal prior to contacting with the enzyme; and

detecting the presence or absence of a D signal,

wherein a decrease in the D signal in the presence of the enzyme as compared to the absence of the enzyme indicates that at least one cell of the cell library expresses a candidate peptide that is a substrate for the enzyme.

2. The method of claim 1, wherein the cell library enriched for expression of peptide display scaffolds is produced by fluorescence activated cell sorting of cells exhibiting the D signal.

3. The method of claim 1, wherein said detecting the presence or absence of the D signal is by fluorescence activated cell sorting.

4. The method of claim 1, wherein C is [A-C_s] wherein A is an allosteric regulator for the enzyme and C_s is a candidate substrate for the enzyme, and

wherein a decrease in the D signal indicates that at least one cell of the cell library expresses a candidate peptide that is a substrate for the enzyme.

5. The method of claim 1, wherein the TM protein is a bacterial outer membrane protein.

6. The method of claim 1, wherein the peptide display scaffold further comprising a linker between C and TM.

7. The method of claim 1, wherein D is an affinity ligand.

22. A method for identifying a peptide substrate for an enzyme, comprising:

contacting a cell expressing a peptide display scaffold according to claim 1 with an enzyme, wherein prior to contacting the cell with the enzyme, the cell exhibits a D₁ signal and does not exhibit a detectable D₂ signal; and

detecting the presence or absence of a D₂ signal,

wherein an increase in the D₂ signal indicates that at least one cell of the cell library expresses a candidate peptide that is a substrate for the enzyme.

23. The method of claim 22, wherein said detecting the presence or absence of the D₂ signal is by fluorescence activated cell sorting.

24. The method of claim 22, wherein an increase in the D₂ signal relative to the D₁ signal in the absence of the enzyme indicates that the cell expresses a candidate peptide that interacts with the enzyme.

25. The method of claim 22, wherein an increase in the D₂ signal relative to the D₁ signal indicates that the cell expresses a candidate peptide that is a substrate for the enzyme.

26. The method of claim 22, wherein C is [A-C_s] wherein A is an allosteric regulator for the enzyme and C_s is a candidate substrate for the enzyme, and

wherein an increase in the D₂ signal indicates that the cell expresses a candidate peptide that is a substrate for the enzyme.

8. A method for screening a library of cells presenting candidate peptides in peptide display scaffolds to identify a peptide inhibitor for an enzyme, comprising:

contacting an enzyme with a cell library enriched for expression of peptide display scaffolds, wherein each peptide display scaffold comprises;

wherein

TM is a transmembrane protein;

S is a substrate for the enzyme

C_i is a candidate inhibitor peptide; and

D is a detectable moiety, wherein the cells of the cell library exhibit

a D signal prior to contacting with the enzyme; and

detecting the presence or absence of a D signal,

wherein maintenance of the D signal in the presence of the enzyme as compared to the absence of the enzyme indicates that C_i is an inhibitor for the enzyme.

9. The method of claim 8, wherein the cell library enriched for expression of peptide display scaffolds is produced by fluorescence activated cell sorting of cells exhibiting the D signal.

10. The method of claim 8, wherein said detecting the presence or absence of the D signal is by fluorescence activated cell sorting.

11. The method of claim 8, wherein the peptide display scaffold further comprising a linker between C_i and TM.

12. The method of claim 8, wherein the TM protein is a bacterial outer membrane protein.

13. The method of claim 8, wherein D is an affinity ligand.

14. A peptide display scaffold, comprising:

15. The peptide display scaffold of claim 14, further comprising at least one linker, wherein the linker is between C₁ and TM or D₂ and TM.

16. The peptide display scaffold of claim 14, wherein the TM protein is a bacterial outer membrane protein.

17. The peptide display scaffold of claim 14, wherein D₁ and D₂ are affinity ligands.

18. The peptide display scaffold of claim 14, further comprising a C₂ between TM and D₂, wherein the peptide display scaffold has the formula

19. The peptide display scaffold of claim 18, further comprising at least one linker, wherein the linker is between C₁ and TM or C₂ and TM.

20. The peptide display scaffold of claim 18, wherein the TM protein is a bacterial outer membrane protein.

21. The peptide display scaffold of claim 18, wherein D₁ and D₂ are affinity ligands.

27. A method for identifying a peptide ligand of an enzyme, comprising:

contacting a cell expressing a peptide display scaffold according to claim 18 with an enzyme, wherein when D₁ is present D₂ does not provide a detectable signal;

detecting the presence or absence of a D₁ signal and a D₂ signal,

wherein said detecting indicates whether C₂ is a peptide ligand for the enzyme.

28. The method of claim 27, wherein said detecting the presence or absence of the D₁ signal and the D₂ signal is by fluorescence activated cell sorting.

29. The method of claim 27, wherein when C₁ is a substrate for the enzyme C₂ is a candidate inhibitor for the enzyme, a change in the D₂ signal relative to the D₁ signal indicates that C₂ is an inhibitor for the enzyme.

30. The method of claim 27, wherein when C₁ is an allosteric regulator for the enzyme and C₂ is a candidate substrate, a change in the D₂ signal relative to the D₁ signal indicates that C₂ is a substrate for the enzyme.

37. A nucleic acid molecule encoding a peptide display scaffold according to claim 14.

38. The nucleic acid molecule of claim 37, further comprising at least one restriction enzyme site between D₁ and C and at least one restriction site between TM and D₂.

39. An expression vector encoding a peptide display scaffold according to claim 14.

40. A host cell comprising a nucleic acid molecule encoding a peptide display scaffold according to claim 14.

41. A kit comprising an expression vector encoding a peptide display scaffold according to claim 14.

42. A nucleic acid molecule encoding a peptide display scaffold according to claim 18.

43. The nucleic acid molecule of claim 42, further comprising at least one restriction enzyme site between D₁ and C₁ and at least one restriction site between C₂ and D₂.

44. An expression vector encoding a peptide display scaffold according to claim 18.

45. A host cell comprising a nucleic acid molecule encoding a peptide display scaffold according to claim 18.

46. A kit comprising an expression vector encoding a peptide display scaffold according to claim 18.

31. 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;

wherein

TM is a transmembrane protein;

S is a substrate for the enzyme

C_i is a candidate inhibitor peptide; and

D₁ and D₂ are first and second detectable moieties, wherein D₁ and

D₂ are different, and wherein prior to contacting the cell with the enzyme,

the cell exhibits a D₁ and does not exhibit a detectable D₂ signal; and

detecting the presence or absence of a D₁ signal and a D₂ signal,

wherein maintenance of the D₁ signal relative to the D₂ signal indicates that C_i is an inhibitor for the enzyme.

32. The method of claim 31, wherein the cell library enriched for expression of peptide display scaffolds is produced by fluorescence activated cell sorting of cells exhibiting the D₁ signal.

33. The method of claim 31, wherein said detecting the presence or absence of the D₁ signal and the D₂ signal is by fluorescence activated cell sorting.

34. The method of claim 31, wherein the peptide display scaffold further comprising a linker between C and TM.

35. The method of claim 31, wherein the TM protein is a bacterial outer membrane protein.

36. The method of claim 31, wherein D₁ and D₂ are affinity ligands.