# US 11,078,479 B2 · Polypeptide display libraries and methods of making and using thereof

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- Number: US 11,078,479 B2
- Document ID: US11078479B2
- Status: Granted
- Kind: B2
- Application number: 16/710,909
- Filed: 2019-12-11
- Published: 2020-10-08
- Granted: 2021-08-03
- Earliest claimed: 2003-08-18
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Patrick S. Daugherty; Paul H. Bessette; Jeffrey Rice
- Web page: https://patentgrep.com/patent/US11078479B2

## CPC

- C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display}
- C07K 14/245: Escherichia (G)
- C12N 15/1044: {Preparation or screening of libraries displayed on scaffold proteins}
- G01N 33/554: the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
- C40B 30/04: by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
- C40B 40/10: Libraries containing peptides or polypeptides, or derivatives thereof
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## 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

### Claim 1

A nucleic acid encoding a fusion protein, the fusion protein comprising: an outer membrane protein and a passenger polypeptide,
wherein the outer membrane protein is a circularly permuted variant of a native outer membrane protein,
wherein a native N-terminus and a native C-terminus of the native outer membrane protein are joined by a linker,
wherein the outer membrane protein comprises a non-native N-terminus and a non-native C-terminus derived from an extracellular loop of the native outer membrane protein,
wherein the passenger polypeptide is fused to the non-native N-terminus or the non-native C-terminus of the outer membrane protein, and
wherein the native outer membrane protein is a bacterial outer membrane protein is OmpA or OmpX.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

The nucleic acid of claim 1, wherein the passenger polypeptide comprises a peptide ligand.

### Claim 4 (depends on 1)

The nucleic acid of claim 1, wherein the fusion protein comprises a label.

### Claim 5 (depends on 4)

The nucleic acid of claim 4, the label comprises a fluorescent agent.

### Claim 6 (depends on 4)

The nucleic acid of claim 4, wherein the label comprises an affinity tag.

### Claim 7 (depends on 4)

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

### Claim 8 (depends on 4)

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

### Claim 9 (depends on 1)

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

### Claim 10 (depends on 1)

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

### Claim 11 (depends on 1)

A host cell comprising an expression vector,
wherein the expression vector comprises the nucleic acid of claim 1,
wherein the host cell is capable of expressing the fusion protein encoded by the nucleic acid and disposing the fusion protein in the outer membrane of the host cell.

### Claim 12 (depends on 11)

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

### Claim 13 (depends on 11)

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

### Claim 14 (depends on 11)

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

### Claim 15 (depends on 11)

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

### Claim 16 (depends on 11)

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

### Claim 17 (depends on 16)

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

### Claim 18 (depends on 17)

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