# US 2010/0167342 A1 · Method of producing biologically active polypeptide having insulinotropic activity

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- Number: US 2010/0167342 A1
- Document ID: US20100167342A1
- Status: Published application
- Kind: A1
- Application number: 12/305,460
- Filed: 2007-06-20
- Published: 2010-07-01
- Earliest claimed: 2006-06-21
- Assignees: Biocon Limited
- Inventors: Ramakrishnan Melarkode; Akundi Venkata Sriram; Kedarnath Nanjund Sastry; Lakshmi Prabha Varadarajalu; Shrikumar Suryanarayan
- Web page: https://patentgrep.com/patent/US20100167342A1

## CPC

- C07K 14/46: from vertebrates
- 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

The present invention relates to a method of producing a biologically active polypeptide having insulinotropic activity, the method comprising steps of: (a) transforming a genetically modified host cell that has protease gene knockout, with a polynucleotide vector encoding the polypeptide; and (b) growing the transformed host cell to produce the biologically active polypeptide; and a method of producing a biologically active polypeptide having an N-terminal recognition site His-Gly with insulinotropic activity, the method comprising steps of: (a) transforming a genetically modified Pichia pastoris that has protease gene STE13 knockout, with a polynucleotide vector encoding the polypeptide; and (b) growing the transformed Pichia pastoris to produce the biologically active polypeptide.

## Claims

### Claim 1

A method of producing a biologically active polypeptide having insulinotropic activity, the method comprising steps of:
a) transforming a genetically modified host cell that has protease gene knockout, with a polynucleotide vector encoding the polypeptide; and
b) growing the transformed host cell to produce the biologically active polypeptide.

### Claim 2 (depends on 1)

The method as claimed in claim 1 further comprising step of isolating and thereafter alpha-amidating the isolated polypeptide.

### Claim 3 (depends on 1)

The method as claimed in claim 1, wherein the host cell is yeast cell.

### Claim 4 (depends on 3)

The method as claimed in claim 3, wherein the yeast cell is Pichia pastoris.

### Claim 5 (depends on 1)

The method as claimed in claim 1, wherein the protease gene is STE13 gene.

### Claim 6 (depends on 1)

The method as claimed in claim 1, wherein the protease gene is an N-terminal dipeptidyl peptidase gene.

### Claim 7 (depends on 1)

The method as claimed in claim 1, wherein growing the transformed cells under conditions which induce expression of the polypeptide.

### Claim 8

A method of producing a biologically active polypeptide having an N-terminal recognition site His-Gly with insulinotropic activity, the method comprising steps of:
a. transforming a genetically modified Pichia pastoris that has protease gene STE13 knockout, with a polynucleotide vector encoding the polypeptide; and
b. growing the transformed Pichia pastoris to produce the biologically active polypeptide.

### Claim 9 (depends on 8)

The method as claimed in claim 8, wherein the polypeptide is Glyexendin-4 of SEQ ID NO 4.

### Claim 10 (depends on 9)

The method as claimed in claim 9, wherein the Glyexendin-4 has corresponding polynucleotide sequence of SEQ ID NO 5.

### Claim 11 (depends on 2)

The method as claimed in claim 2, wherein contacting the glyexedin-4 polypeptide with a C-terminal alpha-amidating enzyme to yield an alpha-amidated exendin-4 polypeptide.

### Claim 12 (depends on 11)

The method as claimed in claim 11, wherein the alpha-amidated exendin-4 polypeptide is HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2.