# US 7,612,019 B2 · Polypeptide display libraries and methods of making and using thereof

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- Number: US 7,612,019 B2
- Document ID: US7612019B2
- Status: Granted
- Kind: B2
- Application number: 11/612,757
- Filed: 2006-12-19
- Published: 2007-05-03
- Granted: 2009-11-03
- Earliest claimed: 2003-08-18
- Assignees: The Regents of the University of California
- Inventors: Patrick S. Daugherty; Paul H. Bessette; Jeffrey Rice
- Web page: https://patentgrep.com/patent/US7612019B2

## 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

An assay method for detecting a target in a sample, said method comprising:
contacting a passenger polypeptide with a sample suspected of containing said target, wherein said passenger polypeptide is displayed on the surface of a biological entity, and wherein said passenger polypeptide is fused to an extracellular terminus of a circularly permuted bacterial outer membrane protein; and detecting a specific interaction between the passenger polypeptide and the target when present, wherein presence of the specific interaction is indicative of the presence of the target in the sample.

### Claim 2 (depends on 1)

The assay of claim 1, wherein the bacterial outer membrane protein is OmpA or OmpX.

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 3)

The assay of claim 3, wherein the target is the enzyme.

### Claim 5 (depends on 1)

The assay of claim 1, wherein the passenger polypeptide has a label.

### Claim 6 (depends on 5)

The assay of claim 5, wherein the label is a fluorescent agent.

### Claim 7 (depends on 5)

The assay of claim 5, wherein the passenger polypeptide has a C-terminal label.

### Claim 8 (depends on 5)

The assay of claim 5, wherein the passenger polypeptide has an N-terminal label.

### Claim 9 (depends on 5)

The assay of claim 5, wherein loss of the label indicates interaction between the passenger polypeptide and the target.

### Claim 10 (depends on 1)

The assay of claim 1, wherein the assay further comprises quantifying the biological entity by flow cytometry, fluorescence, or microscopy.

### Claim 11 (depends on 1)

The assay of claim 1, wherein the biological entity expresses a fluorescent molecule or a luminescent reporter molecule.

### Claim 12 (depends on 1)

The assay of claim 1, wherein the target is associated with a second biological entity or a particle.

### Claim 13 (depends on 12)

The assay of claim 12, wherein interaction between the passenger polypeptide and the target results in a complex between the biological entity and the second biological entity or the particle.

### Claim 14 (depends on 13)

The assay of claim 13, wherein the complex is quantified by flow cytometry, fluorescence, or microscopy.

### Claim 15 (depends on 1)

The assay of claim 1, wherein the passenger polypeptide comprises SEQ ID NO:315.

### Claim 16 (depends on 15)

The assay of claim 15, wherein the passenger polypeptide is capable of binding C-reactive protein.

### Claim 17 (depends on 1)

The assay of claim 1, wherein the passenger polypeptide comprises SEQ ID NO:99, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ TD NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, or SEQ ID NO:256.

### Claim 18 (depends on 1)

The assay of claim 1, wherein the passenger polypeptide consists of SEQ ID NO:99, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, or SEQ ID NO:256.