# US 7,410,775 B2 · Method for making recombinant peptides or proteins using soluble endoproteases

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 7,410,775 B2
- Document ID: US7410775B2
- Status: Granted
- Kind: B2
- Application number: 11/804,557
- Filed: 2007-05-18
- Published: 2007-12-27
- Granted: 2008-08-12
- Earliest claimed: 2000-11-16
- Assignees: Bio-Ker S.R.L.
- Inventors: Marco Vanoni; Paolo Tortora; Giancarlo Tonon; Geoffrey Taylor; Gaetano Orsini
- Web page: https://patentgrep.com/patent/US7410775B2

## CPC

- C12N 9/60: from yeast
- C12P 21/06: produced by the hydrolysis of a peptide bond, e.g. hydrolysate products (preparing foodstuffs by protein hydrolysis A23J3/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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 the expression and secretion in Saccharomyces cerevisiae of readily purifiable soluble variants of the Kex1 endopeptidase of Kluyveromyces lactis and the purification and use thereof for the in vitro processing of recombinant proteins usable in industrial applications. The soluble Kex1 endoproteases described here are free from the transmembrane domain of the native enzyme; the deletion of the transmembrane domain is achieved by removing at least 57 amino acid residues from the C-terminal.

## Claims

### Claim 1

A method for making a recombinant peptide or protein comprising:
(a) producing a fusion protein comprising, at the NH2-terminal end of a peptide or protein of interest, an amino acid sequence containing a dipeptide site hydrolysable by a soluble Kluyveromyces lactis Kex1 endoprotease free from its transmembrane domain;
(b) incubating the fusion protein of step (a) in the presence of a soluble Kluyveromyces lactis Kex1 endoprotease consisting of the amino acid sequence set forth as amino acid residues 103 through 600 of SEQ ID NO 3, or amino acid residues 103 through 611 of SEQ ID NO 3; and
(c) separating and purifying the peptide or protein of interest.

### Claim 2 (depends on 1)

The method of claim 1, wherein the hydrolysable dipeptide site is Lys-Arg, Arg-Arg, or Pro-Arg.

### Claim 3 (depends on 1)

The method of claim 1, wherein the peptide or protein of interest is a recombinant proteins, selected from the group consisting of peptide hormones, interleukins, interferons, cytokines, growth factors, and fibrinolytic enzymes.

### Claim 4 (depends on 1)

The method of claim 1, wherein the soluble Kluyveromyces lactis Kex1 endoprotease is immobilised on an insoluble organic or inorganic support.

### Claim 5 (depends on 1)

The method of claim 1, wherein the peptide or protein of interest is an enzyme selected from the group consisting of lipases, hydrolases, nucleases, oxidases, and phosphatases.