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

• US 9,121,828 B2
• Filed: 09/28/2012
• Issued: 09/01/2015
• 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:
  US13/631,206
• Publication Number:
  US 20130123141A1
• Time in Patent Office: 1,068 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 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:

[D₁-C₁]-TM-[D₂]

wherein TM is a circularly permuted OmpA or OmpX protein;

C₁ comprises a candidate peptide; and

D₁ and D₂ are first and second detectable moieties, wherein D₁ is a heterologous peptide or polypeptide and D₂ is a heterologous peptide or polypeptide, wherein D₁ and D₂ are not the same and wherein D₁-C₁ and D₂ 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 D₂ and TM.

3. The cell of claim 1, wherein the fusion protein comprises a linker between C₁ and TM and a linker between D₂ and TM.

4. The cell of claim 1, wherein detectable moieties D₁ and D₂ are first and second affinity tags which are not the same.

5. The cell of claim 1, wherein C₁ 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 D₁ and D₂ is a fluorescent protein.

7. The cell of claim 1, wherein D₁-C₁ is located at an N-terminal domain of the fusion protein and D₂ is located at a C-terminal domain of the fusion protein.

8. The cell of claim 1, wherein C₁-D₁ is located at a C-terminal domain of the fusion protein and D₂ is located at an N-terminal domain of the fusion protein.

9. The cell 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-C₁ and C₂-D₂ 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 C₁ and TM or between C₂ and TM.

11. The cell of claim 9, wherein the fusion protein comprises a linker between C₁ and TM and a linker between C₂ and TM.

12. The cell of claim 9, wherein detectable moieties D₁ and D₂ are first and second affinity tags which are not the same.

13. The cell of claim 9, wherein C₁ 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 D₁ and D₂ is a fluorescent protein.

15. The cell of claim 9, wherein D₁-C₁ is located at an N-terminal domain of the fusion protein and C₂-D₂ is located at a C-terminal domain of the fusion protein.

16. The cell of claim 9, wherein C₁-D₁ is located at a C-terminal domain of the fusion protein and D₂-C₂ 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 D₂ signal from detectable moiety D₂ relative to a D₁ signal from detectable moiety D₁, wherein an increase in the D₂ signal relative to the D₁ signal indicates that C₁ 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 C₁ comprises a substrate for the enzyme and C₂ comprises a candidate inhibitor for the enzyme; and

detecting the presence or absence of a D₁ signal and a D₂ signal, wherein when C₁ comprises a substrate for the enzyme and C₂ comprises a candidate inhibitor for the enzyme, maintenance of the D₁ signal relative to the D₂ signal following contact with the enzyme indicates that C₂ 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 C₁ comprises an allosteric regulator for the enzyme and C₂ comprises a candidate substrate for the enzyme; and

determining whether there is a change in a D₂ signal from detectable moiety D₂ relative to a D₁ signal from detectable moiety D₁, wherein a change in the D₂ signal relative to the D₁ signal indicates that C₂ 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 C₁ comprises a known substrate for the enzyme and C₂ comprises a candidate allosteric regulator for the enzyme; and

determining whether there is a change in a D₂ signal from detectable moiety D₂ relative to a D₁ signal from detectable moiety D₁, wherein a change in the D₂ signal relative to the D₁ signal indicates that C₂ comprises an allosteric regulator for the enzyme.