# US 10,501,506 B2 · Polypeptide, and use thereof

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- Number: US 10,501,506 B2
- Document ID: US10501506B2
- Status: Granted
- Kind: B2
- Application number: 14/901,005
- Filed: 2014-06-25
- Published: 2016-06-02
- Granted: 2019-12-10
- Earliest claimed: 2013-06-26
- Assignees: Kaneka Corporation; National Institute of Advanced Industrial Science and Technology
- Applicants: Kaneka Corporation; National Institute of Advanced Industrial Science and Technology
- Inventors: Teruyuki Nishi; Kiyoshi Asai; Hiroko Hagiwara; Masayuki Machida
- Web page: https://patentgrep.com/patent/US10501506B2

## CPC

- C07K 14/39: from yeasts
- C12N 15/815: {for yeasts other than Saccharomyces}
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- 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)
- 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

This invention provides a novel means that allows efficient secretion and production of a target protein in a host cell. This invention concerns a novel polypeptide having an activity of improving the secretion productivity of a target protein, a gene comprising a nucleotide sequence encoding such polypeptide, a vector comprising such gene, a transformant obtained by transforming a host cell with such vector, and a method for producing a protein comprising a step of culturing such transformant and recovering a target protein from the culture product.

## Claims

### Claim 1

A method for producing a target protein comprising:
(i) culturing a transformant of a host cell, comprising:
a gene encoding the target protein operably linked to a methanol-inducible promoter, and
a vector which comprises a gene encoding a polypeptide, wherein the polypeptide gene comprises:
(a) a polynucleotide encoding a polypeptide comprising SEQ ID NO: 32,
(b) a polynucleotide encoding a polypeptide comprising an amino acid sequence with 95% or higher sequence identity with SEQ ID NO: 32, or
(c) a polynucleotide encoding a polypeptide comprising SEQ ID NO: 32 with up to 5 substitutions, deletions, insertions and/or additions of amino acids; and
(ii) expressing the polypeptide and expressing the target protein; and
(iii) recovering a target protein from the culture product.

### Claim 2 (depends on 1)

The method of claim 1, wherein the culturing occurs in a culture medium which comprises methanol and/or glycerol, and optionally glucose.

### Claim 3 (depends on 1)

The method of claim 1, wherein a polynucleotide encoding a signal sequence is at the 5′ end of the target protein gene.

### Claim 4 (depends on 1)

The method of claim 1, wherein a host cell is a yeast cell, bacteria cell, fungal cell, insect cell, or animal cell.

### Claim 5 (depends on 4)

The method of claim 4, wherein the host cell is the yeast cell, and the yeast cell is a methanol-assimilating yeast cell.

### Claim 6 (depends on 5)

The method of claim 5, wherein the methanol-assimilating yeast cell belongs to the genus Ogataea or Komagataella.

### Claim 7 (depends on 1)

The method of claim 1, wherein the target protein is a human therapeutic protein.

### Claim 8 (depends on 1)

The method of claim 1, wherein the polypeptide is not naturally expressed in the host cell.

### Claim 9 (depends on 1)

The method of claim 1, wherein the methanol inducible promoter is also a glycerol inducible promoter.

### Claim 10 (depends on 1)

The method of claim 1, wherein the polynucleotide is (a) a polynucleotide encoding a polypeptide comprising SEQ ID NO: 32.

### Claim 11 (depends on 1)

The method of claim 1, wherein the polynucleotide is (b) a polynucleotide encoding a polypeptide comprising an amino acid sequence with 95% or higher sequence identity with SEQ ID NO: 32.

### Claim 12 (depends on 1)

The method of claim 1, wherein the polynucleotide is (c) a polynucleotide encoding a polypeptide comprising SEQ ID NO: 32 with up to 5 substitutions, deletions, insertions and/or additions of amino acids.