# US 10,308,679 B2 · Soluble intein fusion proteins and methods for purifying biomolecules

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- Number: US 10,308,679 B2
- Document ID: US10308679B2
- Status: Granted
- Kind: B2
- Application number: 15/511,908
- Filed: 2015-10-23
- Published: 2017-10-12
- Granted: 2019-06-04
- Earliest claimed: 2014-11-03
- Assignees: Merck Patent GmbH
- Applicants: Merck Patent GmbH
- Inventors: Martin Zillmann; Joe Orlando
- Web page: https://patentgrep.com/patent/US10308679B2

## CPC

- C12N 9/52: derived from bacteria {or Archaea}
- C07K 1/22: Affinity chromatography or related techniques based upon selective absorption processes
- C07K 14/00: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C12N 9/93: {Ligases (6)}
- C12N 11/082: obtained by reactions only involving carbon-to-carbon unsaturated bonds
- C12N 11/14: Enzymes or microbial cells immobilised on or in an inorganic carrier
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C07K 2319/92: containing an intein ("protein splicing")domain
- 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)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

The present invention relates to fusion proteins comprising an N-intein polypeptide and an N-intein solubilization partner, and affinity chromatography matrices comprising such fusion proteins, as well as methods of using same.

## Claims

### Claim 1

A fusion protein comprising an N-intein polypeptide and an N-intein solubilization partner polypeptide joined by a peptide bond, wherein the N-intein solubilization partner polypeptide has a molecular weight of less than about 15 kDa, an Aliphatic Index value less than about 60, and a Grand Average Hydropathy value less than −1, and enhances solubility of the N-intein polypeptide compared to the N-intein polypeptide expressed in the absence of the solubilization partner polypeptide; and maintains the catalytic activity of the N-intein polypeptide when complexed with a C-intein domain, the C-intein domain not being a part of the fusion protein.

### Claim 2 (depends on 1)

The fusion protein of claim 1, wherein the N-intein polypeptide is GP41-1 N-intein (SEQ ID NO:1), or a variant thereof.

### Claim 3 (depends on 2)

The fusion protein of claim 2, wherein the variant of GP41-1 N-intein has an amino acid other than cysteine residue at positions 7, 65 and 89 of SEQ ID NO:1.

### Claim 4 (depends on 3)

The fusion protein of claim 3, wherein the variant of GP41-1 N-intein has an alanine at position 7, a threonine residue or an alanine residue at position 65 and either a methionine residue, lysine residue or an asparagine residue at position 89 of SEQ ID NO:1.

### Claim 5 (depends on 1)

The fusion protein of claim 1, wherein the variant of GP41-1 N-intein has an amino acid sequence having at least about 90% identity to GP41-1 (SEQ ID NO:1).

### Claim 6 (depends on 1)

The fusion protein of claim 1, wherein the fusion protein is covalently attached to a solid support.

### Claim 7 (depends on 6)

The fusion protein of claim 6, wherein covalent attachment of the fusion protein to a solid support is through an amino acid of the N-intein solubilization partner polypeptide.

### Claim 8 (depends on 7)

The fusion protein of claim 7, wherein the amino acid is cysteine.

### Claim 9 (depends on 1)

The fusion protein of claim 1, wherein the N-intein polypeptide comprises a sequence selected from SEQ ID NOs:1-8 and 29-56.

### Claim 10 (depends on 1)

The fusion protein of claim 1, wherein the N-intein solubilization partner polypeptide is joined to the N-terminal end of the N-intein polypeptide.

### Claim 11 (depends on 1)

The fusion protein of claim 1, wherein the N-intein solubilization partner polypeptide is joined to the C-terminal end of the N-intein polypeptide.

### Claim 12 (depends on 1)

The fusion protein of claim 1, wherein the N-intein solubilization partner polypeptide is N-intein solubilization partner 138 (SEQ ID NO:15), or a variant thereof.

### Claim 13 (depends on 12)

The fusion protein of claim 12, wherein the variant of N-intein solubilization partner 138 comprises SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.

### Claim 14 (depends on 1)

The fusion protein of claim 1, wherein the fusion protein is produced in E. coli under conditions in which less than 25% by mass of the fusion protein produced is present in inclusion bodies.

### Claim 15 (depends on 1)

The fusion protein of claim 1, wherein the fusion protein is modified to include a detectable label.

### Claim 16 (depends on 15)

The fusion protein of claim 15, wherein the detectable label is a fluorescent dye.

### Claim 17 (depends on 1)

The fusion protein of claim 1, wherein the fusion protein is expressed in Escherichia coli, Corynebacterium glutamicum, Pseudomonas fluorescens, Lactococcus lactis, Pichia pastoris, Saccharomyces cerevisiae, Zea maize, Nicotinia tabacum, Daucus carota, SF9 cells, CHO cells, NS0 cells, or HEK 293 cells.

### Claim 18 (depends on 1)

An affinity chromatography matrix comprising the fusion protein of claim 1 attached to a solid support.

### Claim 19 (depends on 18)

The affinity chromatography matrix of claim 18, wherein the solid support is a chromatography resin.

### Claim 20 (depends on 19)

The affinity chromatography matrix of claim 19, wherein the chromatography resin includes a hydrophilic polyvinyl ether base.

### Claim 21 (depends on 18)

The affinity chromatography matrix of claim 18, wherein the solid support is a bead, a hollow fiber, a solid fiber, a pad, a gel, a membrane, a cassette, a column, a chip, a slide, a plate or a monolith.

### Claim 22 (depends on 21)

The affinity chromatography matrix of claim 21, wherein the solid support is a magnetic bead.

### Claim 23 (depends on 18)

The affinity chromatography matrix of claim 18, wherein the solid support comprises controlled pore glass, silica, zirconium oxide, titanium oxide, agarose, polymethacrylate, polyacrylate, polyacrylamide, polyvinyl alcohol, polystyrene or derivatives thereof.

### Claim 24 (depends on 18)

The affinity chromatography matrix of claim 18, wherein the matrix further comprises a spacer molecule between the fusion protein and solid support.

### Claim 25 (depends on 18)

The affinity chromatography matrix of claim 18, wherein the fusion protein is attached to the solid support at a single site in the N-intein solubilization partner polypeptide.

### Claim 26 (depends on 18)

The affinity chromatography matrix of claim 18, wherein the fusion protein is attached to the solid support at more than one site in the N-intein solubilization partner polypeptide.

### Claim 27 (depends on 18)

The affinity chromatography matrix of claim 18, wherein the N-intein polypeptide in the fusion protein remains active when the fusion protein is attached to the solid support.

### Claim 28 (depends on 18)

The affinity chromatography matrix of claim 18, wherein the N-intein polypeptide in the fusion protein is oriented away from the solid support when the fusion protein is attached to the solid support.

### Claim 29 (depends on 1)

A method of affinity purifying a target molecule in a sample, the method comprising:
a) providing a sample containing a first fusion protein comprising a C-intein polypeptide joined to a target molecule by a peptide bond;
b) contacting the sample with an affinity chromatography matrix that comprises a second fusion protein, wherein the second fusion protein is the fusion protein of claim 1, under conditions in which the C-intein polypeptide in the first fusion protein selectively binds to the N-intein polypeptide in the second fusion protein to form an intein complex that is inactive;
c) washing the affinity chromatography matrix containing the inactive intein complex to remove unbound contaminants;
d) exposing the intein complex to conditions under which the intein complex is active and cleaves the target molecule from the C-intein polypeptide; and
e) recovering the cleaved target molecule.

### Claim 30 (depends on 29)

The method of claim 29, wherein the sample is a crude protein preparation.

### Claim 31 (depends on 29)

The method of claim 29, wherein the target molecule is a monoclonal antibody to a therapeutic target.

### Claim 32 (depends on 29)

The method of claim 29, wherein the target molecule is a therapeutic target.

### Claim 33 (depends on 29)

The method of claim 29, wherein the conditions under which the C-intein polypeptide in the first fusion protein selectively binds to the N-intein polypeptide in the second fusion protein to form a catalytically-inactive intein complex include:
a) a temperature in the range of about 4-25° C., and a buffer comprising 100 mM Tris-HCl, 25 mM NaCl, 0.1 mM zinc chloride, pH=9;
b) a temperature in the range of about 4-25° C., and a buffer comprising 50 mM NaAc, 0.5 M NaCl, pH=5; or
c) a temperature in the range of about 4-25° C., and a buffer comprising 0.5 M NaCl, 10 mM Tris-HCl, pH=8.

### Claim 34 (depends on 29)

The method of claim 29, wherein the conditions that promote catalytic activity of the intein complex include:
a) a buffer comprising 50 mM Tris-HCl, pH=7.0, 300 mM NaCl, 1 mM EDTA;
b) a buffer comprising 0.3 M L-arginine, 5 mM EDTA, 50 mM phosphate buffer, pH=6.5; or
c) a buffer comprising 0.5 M NaCl, 10 mM Tris-HCl, 50 mM DTT, pH=8.0.

### Claim 35 (depends on 29)

The method of claim 29, wherein the N-intein solubilization partner polypeptide is N-intein solubilization partner 138 (SEQ ID NO:15) or a variant thereof.

### Claim 36 (depends on 29)

The method of claim 29, wherein the N-intein polypeptide is GP41-1 N-intein (SEQ ID NO:1) or a variant thereof, and the C-intein polypeptide is GP41-1 C-intein (SEQ ID NO:9).

### Claim 37 (depends on 29)

The method of claim 29, further comprising cleaning, regenerating or storing the affinity chromatography matrix for subsequent use.

### Claim 38 (depends on 1)

A method of screening for an intein complex that is suitable for use in affinity purification, the method comprising:
a) contacting a first fusion protein that comprises a C-intein polypeptide joined to a target molecule by a peptide bond with a second fusion protein, wherein the second fusion protein is the fusion protein of claim 1, under conditions in which the C-intein polypeptide in the first fusion protein selectively binds to the N-intein polypeptide in the second fusion protein to form an intein complex; and
determining whether the target molecule is cleaved from the C-intein polypeptide under conditions which support intein activity, wherein the presence of the cleaved target molecule is indicative of a catalytically-active intein complex.