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

• US 7,666,817 B2
• Filed: 08/30/2006
• Issued: 02/23/2010
• 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: Boulware, Kevin T., Daugherty, Patrick Sean
• Primary Examiner(s): WESSENDORF, TERESA D
• Application Number:
  US11/514,377
• Publication Number:
  US 20070065878A1
• Time in Patent Office: 1,273 Days
• Field of Search: 506 1- 4, 506/14, 506/18, 435/7.2, 435/7.37, 435/7.44
• US Class Current: 506/4
• 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 identifying a peptide substrate for an enzyme, comprising:

contacting a cell with an enzyme, the cell comprising a cell outer membrane and expressing a peptide display scaffold, the peptide display scaffold comprising

a fusion protein comprising the formula;

2. The method of claim 1, wherein said detecting is by fluorescence activated cell sorting.

3. The method of claim 1, wherein an increase in the detectable D₂ signal relative to the detectable D₁ signal identifies the member of the library of candidate peptide substrates as a peptide substrate for the enzyme.

4. The method of claim 1, wherein C₁ is [A-C_s] wherein A is an allosteric regulator for the enzyme and C_s is a member of a library of candidate peptide substrates for the enzyme, and

wherein the presence of a detectable D₂ signal above a background level of a detectable D₂ signal identifies the member of the library of candidate peptide substrates as a peptide substrate for the enzyme.

10. The method of claim 1, wherein the fusion protein further comprises at least one linker, wherein the linker is between C₁ and TM or D₂ and TM.

11. The method of claim 1, wherein detectable moieties D₁ and D₂ are affinity tags.

12. The method of claim 1, wherein D₁-C₁ is located at an N-terminal domain of said fusion protein and D₂ is located at a C-terminal domain of said fusion protein.

13. The method of claim 1, wherein D₁-C₁ is located at a C-terminal domain of said fusion protein and D₂ is located at an N-terminal domain of said fusion protein.

5. 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 a fusion protein comprising the formula;

6. The method of claim 5, wherein the cell is a member of a cell library enriched for expression of the peptide display scaffold by fluorescence activated cell sorting of cells exhibiting the D₁ signal.

7. The method of claim 5, wherein said detecting the presence or absence of the D₁ signal and the D₂ signal is by fluorescence activated cell sorting.

8. The method of claim 5, wherein the peptide display scaffold further comprising a linker between C_i and TM.

9. The method of claim 5, wherein D₁ and D₂ are affinity ligands.