Polypeptide display libraries and methods of making and using thereof

• US 10,041,063 B2
• Filed: 08/07/2015
• Issued: 08/07/2018
• 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): Boesen, Christian C
• Application Number:
  US14/821,580
• Publication Number:
  US 20160032279A1
• Time in Patent Office: 1,096 Days
• Field of Search: None
• 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 method for identifying a target in a sample, the method comprising:

(a) contacting the sample with a fusion protein, wherein the fusion protein comprises an outer membrane protein and a passenger polypeptide, wherein the outer membrane protein is a bacterial outer membrane protein OmpA, OmpX, or OmpT;

(i) wherein a native N-terminus and a native C-terminus of the outer membrane protein are joined by a linker,

(ii) wherein the outer membrane protein comprises a non-native N-terminus and a non-native C-terminus derived from an extracellular loop of the outer membrane protein, and

(iii) wherein the passenger polypeptide is fused to the non-native N-terminus or the non-native C-terminus of the outer membrane protein; and

(b) detecting a specific interaction between the passenger polypeptide and the target, wherein the presence of the specific interaction is indicative of the presence of the target in the sample.

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

3. The method of claim 1, wherein the passenger polypeptide comprises a peptide ligand.

4. The method of claim 1, wherein the fusion protein comprises a label.

5. The method of claim 4, wherein the label comprises a fluorescent agent.

6. The method of claim 4, wherein the label is C-terminal to the fusion protein.

7. The method of claim 4, wherein the label is N-terminal to the fusion protein.

8. The method of claim 4, wherein the label comprises an affinity tag.

9. The method of claim 1, wherein the passenger polypeptide is linked or fused directly to the non-native C-terminus of the outer membrane protein.

10. The method of claim 1, wherein the passenger polypeptide is linked or fused directly to the non-native N-terminus of the outer membrane protein.

11. The method of claim 1, wherein the fusion protein is disposed in an outer membrane of a host cell, and wherein at least one of the non-native N-terminus and the non-native C-terminus is positioned extracellularly.

12. The method of claim 11, wherein the non-native N-terminus is positioned extracellularly.

13. The method of claim 11, wherein the non-native C-terminus is positioned extracellularly.

14. The method of claim 11, wherein both the non-native C-terminus and the non-native N-terminus are positioned extracellularly.

15. The method of claim 11, wherein the host cell is a bacterial cell, a yeast cell or a mammalian cell.

16. The method of claim 11, wherein the host cell is a bacterial cell.

17. The method of claim 16, wherein the bacterial cell is an Escherichia coli cell.