Polypeptide display libraries and methods of making and using thereof

• US 8,361,933 B2
• Filed: 09/21/2009
• Issued: 01/29/2013
• Est. Priority Date: 08/18/2003
• 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 S., Bessette, Paul H., Rice, Jeffrey
• Primary Examiner(s): Marschel, Ardin
• Assistant Examiner(s): BOESEN, CHRISTIAN C
• Application Number:
  US12/563,897
• Publication Number:
  US 20100113303A1
• Time in Patent Office: 1,226 Days
• Field of Search: None
• US Class Current: 506/14
• CPC Class Codes: C07K 14/245 Escherichia (G)
• 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: 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 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: G01N 33/554 the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells

Abstract

Disclosed herein are expression vectors which display a passenger polypeptide on the outer surface of a biological entity. As disclosed herein the displayed passenger polypeptide is capable of interacting or binding with a given ligand. Also disclosed are methods of making and using the expression vectors. N/C terminal fusion expression vectors and methods of making and using are also disclosed.

Claims

1. A composition comprising:

a host cell, wherein the host cell is a bacterial cell; and

a fusion protein disposed in an outer membrane of the host cell, the fusion protein comprising

(a) a circularly permuted bacterial outer membrane protein, comprising a bacterial OmpX protein or a bacterial OmpA protein, wherein

(i) the native N-terminus and the native C-terminus of the bacterial OmpX protein or the bacterial OmpA protein are joined by a linker, wherein the linker is a short flexible peptide, and

(ii) a non-native N-terminus and a non-native C-terminus are derived from an extracellular loop of the bacterial OmpX protein or the bacterial OmpA protein, wherein at least one of the non-native N-terminus and the non-native C-terminus is positioned extracellularly, and

(b) a passenger polypeptide linked or fused directly to the non-native N-terminus or the non-native C-terminus of the circularly permuted bacterial outer membrane protein, wherein the passenger polypeptide is displayed on the outer membrane of the host cell.

2. The composition of claim 1, wherein the passenger polypeptide is a substrate for an enzyme.

3. The composition of claim 1, wherein the fusion protein comprises a label.

4. The composition of claim 3, wherein the label is a fluorescent agent.

5. The composition of claim 3, wherein the label is C-terminal to the fusion protein.

6. The composition of claim 3, wherein the label is N-terminal to the fusion protein.

7. The composition of claim 1, wherein the passenger polypeptide is linked or fused directly to the non-native C-terminus of the circularly permuted bacterial outer membrane protein.

8. The composition of claim 1, wherein the passenger polypeptide is linked or fused directly to the non-native N-terminus of the circularly permuted bacterial outer membrane protein.

9. The composition of claim 1, wherein the bacterial cell is an Escherichia coli cell.

16. The composition of claim 1, wherein the passenger polypeptide comprises a peptide ligand.

17. The composition of claim 3, wherein the label comprises an affinity tag.

19. The composition of claim 7, wherein the non-native N-terminus of the circularly permuted bacterial outer membrane protein is exposed on the outer membrane of the host cell.

20. The composition of claim 8, wherein the non-native C-terminus of the circularly permuted bacterial outer membrane protein is exposed on the outer membrane of the host cell.

10. A polypeptide display library comprising:

a plurality of host cells, wherein the host cells are bacterial cells;

each member of the plurality comprising

a fusion protein disposed in an outer membrane of the host cell, the fusion protein comprising

(a) a circularly permuted bacterial outer membrane protein, comprising a bacterial OmpX protein or a bacterial OmpA protein, wherein

(i) the native N-terminus and the native C-terminus of the bacterial OmpX protein or the bacterial OmpA protein are joined by a linker, wherein the linker is a short flexible peptide, and

(ii) a non-native N-terminus and a non-native C-terminus are derived from an extracellular loop of the bacterial OmpX protein or the bacterial OmpA protein, wherein at least one of the non-native N-terminus and the non-native C-terminus is positioned extracellularly, and

(b) a passenger polypeptide linked or fused directly to the non-native N-terminus or the non-native C-terminus of the circularly permuted bacterial outer membrane protein, wherein the passenger polypeptide is displayed on the outer membrane of the host cell.

11. The polypeptide display library of claim 10, wherein the fusion protein comprises a label.

12. The polypeptide display library of claim 11, wherein the label is a fluorescent agent.

13. The polypeptide display library of claim 11, wherein the label is C-terminal to the fusion protein.

14. The polypeptide display library of claim 11, wherein the label is N-terminal to the fusion protein.

15. The polypeptide display library of claim 10, wherein the bacterial cells are Escherichia coli cells.

18. The polypeptide display library of claim 10, wherein the passenger polypeptide comprises a peptide ligand.

21. The polypeptide display library of claim 11, wherein the label comprises an affinity tag.