# US 9,518,084 B2 · Epitope tag for affinity-based applications

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- Number: US 9,518,084 B2
- Document ID: US9518084B2
- Status: Granted
- Kind: B2
- Application number: 13/698,624
- Filed: 2011-05-25
- Published: 2013-05-09
- Granted: 2016-12-13
- Earliest claimed: 2010-05-25
- Assignees: Vib Vzw; Vrije Universiteit Brussel
- Applicants: Els Pardon; Jan Steyaert; Lode Wyns
- Inventors: Els Pardon; Jan Steyaert; Lode Wyns
- Web page: https://patentgrep.com/patent/US9518084B2

## CPC

- C07K 5/1021: {with the first amino acid being acidic}
- C07K 1/22: Affinity chromatography or related techniques based upon selective absorption processes
- C07K 2319/40: containing a tag for immunodetection, or an epitope for immunisation
- 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)

## Abstract

Described is an epitope tag useful in affinity-based applications. The invention further includes fusion proteins, methods for preparing fusion proteins, nucleic acid molecules encoding these fusion proteins and recombinant host cells that contain these nucleic acid molecules. The invention also relates to nanobodies and other affinity ligands specifically recognizing the epitope tag, and uses thereof in affinity-based applications.

## Claims

### Claim 1

A method for purifying a non-naturally occurring fusion protein comprising (i) an epitope tag consisting of SEQ ID NO:1 and (ii) a target protein, the method comprising:
a) applying a solution containing the non-naturally occurring fusion protein to a solid support possessing an immobilized affinity ligand that binds to an epitope tag consisting of SEQ ID NO:1, wherein the solid support is a column or a bead, and wherein the affinity ligand is an antibody, antibody fragment, or single-domain antibody;
b) forming a complex between the immobilized affinity ligand and the non-naturally occurring fusion protein;
c) removing weakly bound or unbound molecules; and
d) eluting bound molecules.

### Claim 2 (depends on 1)

The method of claim 1, wherein the solid support is a bead.

### Claim 3

A method for immobilizing and/or capturing a non-naturally occurring fusion protein on a solid support, wherein the non-naturally occurring fusion protein comprises (i) an epitope tag consisting of SEQ ID NO:1 and (ii) a target protein, the method comprising:
a) applying a solution containing a non-naturally occurring fusion protein to a solid support possessing an immobilized affinity ligand that binds to an epitope tag consisting of SEQ ID NO:1, wherein the solid support is a column or a bead, and wherein the affinity ligand is an antibody, antibody fragment, or single-domain antibody;
b) forming a complex between the immobilized affinity ligand and the fusion protein; and
c) removing weakly bound or unbound molecules.

### Claim 4 (depends on 3)

The method of claim 3, wherein the solid support is a bead.

### Claim 5 (depends on 3)

The method of claim 3, wherein the solid support is a column.

### Claim 6 (depends on 1)

The method of claim 1, wherein the solid support is a column.