Method for producing interleukin-2 protein using methylotrophic yeast

Patent metadata

Field	Value
Publication number	US-2018237488-A1
Application number	US-201515523290-A
Country	US
Kind code	A1
Filing date	May 13, 2015
Priority date	Oct 30, 2014
Publication date	Aug 23, 2018
Grant date	—

Abstract

The present invention relates to a method for producing an interleukin-2 protein using methylotrophic yeast. The method for producing interleukin-2 according to the present invention shows high cell growth and protein synthesis rates by use of the established optimal cell line, and produces a large amount of a protein comprising interleukin-2 by use of the established optimal culture conditions utilizing methanol that is an inexpensive carbon source. In addition, the method according to the present invention isolates and purifies the protein by a simple process. Accordingly, the method according to the present invention highly pure interleukin-2, and thus has a significant effect on the mass-production of interleukin-2.

First claim

1 . A method for producing interleukin-2, comprising the steps of:
(a) cloning an interleukin-2 expression construct for yeast, wherein the expression construct comprises a methanol oxidase (MOX) promoter, a human serum albumin gene or a fragment thereof, and an interleukin-2 gene; (b) transforming yeast host cells with the expression construct prepared in step (a), and culturing the transformed yeast cells to express interleukin-2; and (c) isolating the expressed interleukin-2 from the transformed yeast cells cultured in step (b).

2 . The method of claim 1 , wherein the expression construct in step (a) further comprises a tobacco etch virus protease site.

3 . The method of claim 1 , wherein the yeast host in step (b) is any one selected from among Hansenula polymorpha, Pichia pastoris, Candia boidini, Pichia methanolica , and Ogataea minuta.

4 . The method of claim 1 , wherein the culturing in step (b) is performed in YPM (2% (w/v) bacto-peptone, 1% (w/v) bacto-yeast extract, 3% (w/v) methanol) medium.

5 . The method of claim 4 , wherein the culturing in step (b) is performed under the following conditions: a methanol concentration of 1% (w/v) to 10% (w/v), a culture temperature of 25° C. to 45° C., a culture pH of 4.5 to 7.0, and a shaking speed of 100 to 300 rpm.

6 . The method of claim 1 , wherein the methanol oxidase (MOX) promoter has a nucleotide sequence of SEQ ID NO:1; the human serum albumin gene or the fragment thereof has a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:3; and the interleukin-2 gene has a nucleotide sequence of SEQ ID NO:4.

7 . A method for producing interleukin-2, comprising the steps of:
(a) culturing Hansenula polymorpha transformed with an interleukin-2 expression construct for yeast, wherein the expression construct comprises a methanol oxidase (MOX) promoter, a human serum albumin gene or a fragment thereof, a tobacco etch virus protease site, and an interleukin-2 gene; (b) isolating a protein from the culture of step (a); and (c) treating the isolated protein of step (b) with tobacco etch virus protease to separate interleukin-2.

8 . The method of claim 7 , wherein the transformed Hansenula polymorpha in step (a) is a strain deposited under accession number Hansenula polymorpha KCTC18329P.

9 . The method of claim 7 , wherein the culturing in step (a) is performed in YPM (2% (w/v) bacto-peptone, 1% (w/v) bacto-yeast extract, 3% (w/v) methanol) medium.

10 . The method of claim 9 , wherein the culturing in step (a) is performed under the following conditions: a methanol concentration of 1% (w/v) to 10% (w/v), a culture temperature of 25° C. to 45° C., a culture pH of 4.5 to 7.0, and a shaking speed of 100 to 300 rpm.

11 . The method of claim 7 , wherein the treating with the tobacco etch virus protease is performed at a temperature of 28° C. to 32° C. for 4 to 8 hours.

Assignees

• Univ Soonchunhyang Ind Acad Coop Found

Inventors

• Rhee Sang-Ki
• Park Eun O
• Seo Hoon
• Choi Guang Jin
• Song Keon-Hyoung

Classifications

• C07K14/765

  Serum albumin, e.g. HSA · CPC title

• C12N15/81 Primary

  for yeasts · CPC title

• C07K14/55 Primary

  IL-2 · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C07K2319/50

  containing protease site · CPC title