# US 8,465,959 B2 · Proteases and methods for producing them

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- Number: US 8,465,959 B2
- Document ID: US8465959B2
- Status: Granted
- Kind: B2
- Application number: 12/147,683
- Filed: 2008-06-27
- Published: 2008-11-27
- Granted: 2013-06-18
- Earliest claimed: 2003-06-19
- Assignees: Novozymes A/S
- Applicants: Soren Flensted Lassen
- Inventors: Soren Flensted Lassen
- Web page: https://patentgrep.com/patent/US8465959B2

## CPC

- C11D 3/38618: {Protease or amylase in liquid compositions only}
- A23K 20/174: Vitamins
- A23K 20/189: Enzymes
- A23K 20/30: {Oligoelements}
- C11D 3/38609: {Protease or amylase in solid compositions only}
- C12N 9/58: derived from fungi
- C11D: DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- A23K: FODDER
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A secreted mature polypeptide derived from an S2A or S1E protease is described which after maturation has protease activity, which polypeptide when expressed and before maturation comprises a heterologous pro-region. Further, a method for producing polypeptide is described including the step of cultivating a recombinant host cell including a polynucleotide encoding a secreted mature polypeptide which after maturation has protease activity, which polypeptide when expressed and before maturation includes a heterologous pro-region derived from an S2A or S1E protease to produce a supermatant including the polypeptide. The polypeptide may also be recovered.

## Claims

### Claim 1

A method for producing a secreted, mature polypeptide, the method comprising:
(a) cultivating a recombinant host cell comprising a polynucleotide encoding a protein comprising:
(i) a heterologous pro-region comprising a sequence having at least 90% sequence identity to SEQ ID NO:30 and having activity to enhance expression of the protein,
(ii) a signal peptide comprising a sequence encoded by a polynucleotide having at least 90% sequence identity to residues 1-81 of SEQ ID NO:2, and
(iii) an amino acid sequence having at least 92% sequence identity to residues 166-353 of SEQ ID NO:43 and that has protease activity, and
(b) culturing the host cell so as to produce a culture supernatant comprising the secreted, mature polypeptide, said secreted mature polypeptide lacking the heterologous pro-region and the signal peptide, and comprising the sequence having at least 90% sequence identity to residues 166-353 of SEQ ID NO:43 , wherein said sequence has protease activity, and
(c) recovering the secreted, mature polypeptide.

### Claim 2

An isolated polynucleotide encoding a polypeptide comprising;
(i) a heterologous pro-region comprising a sequence having at least 90% sequence identity to SEQ ID NO:30 and having activity to enhance expression of the protein,
(ii) a signal peptide comprising a sequence encoded by a polynucleotide having at least 90% sequence identity to residues 1-81 of SEQ ID NO:2, and
(iii) an amino acid sequence having at least 92% sequence identity to residues 166-353 of SEQ ID NO:43 and that has protease activity.

### Claim 3 (depends on 2)

A recombinant expression vector or polynucleotide construct comprising the polynucleotide of claim 2.

### Claim 4 (depends on 2, 3)

A recombinant host cell comprising the polynucleotide of claim 2, or the expression vector or polynucleotide construct as defined in claim 3.

### Claim 5 (depends on 4)

The recombinant host cell according to claim 4 which is a Bacillus cell.

### Claim 6

A method for producing a secreted mature polypeptide, the method comprising:
(a) cultivating a recombinant host cell comprising a polynucleotide encoding a protein comprising:
(i) a heterologous pro-region comprising a sequence having at least 92% sequence identity to SEQ ID NO:30 and having activity to enhance expression of the protein,
(ii) a signal peptide comprising a sequence encoded by a polynucleotide having at least 90% sequence identity to residues 1-81 of SEQ ID NO:2, and
(iii) an amino acid sequence having at least 98% sequence identity to resides 166-353 of SEQ ID NO:43 and that has protease activity, and
(b) culturing the host cell so as to produce a culture supernatant comprising the secreted, mature polypeptide, said secreted mature polypeptide lacking the heterologous pro-region and the signal peptide, and comprising the sequence having at least 98% sequence identity to residues 166-353 of SEQ ID NO:43, wherein said sequence has protease activity.

### Claim 7 (depends on 6)

The method of claim 6, wherein the secreted mature polypeptide comprises an amino acid sequence having at least 99% sequence identity to residues 166-353 of SEQ ID NO:43.