Producing il-21 in prokaryotic host cells

FIELD: medicine.

SUBSTANCE: claimed are expression vectors for obtaining IL-21 in E.coli cells. IL-21 coding nucleotide sequence, included in composition of novel vectors, contains modifications aimed at optimisation of codons and secondary mRNA structure for translation in E.coli. As a result of transformation with claimed vector structures E.coli strains suitable for industrial scale application have been obtained. Methods of wide scale IL-21 production which use said strains have been elaborated, allowing to obtain more than 1g/l of recombinant cytokine.

EFFECT: novel compounds possess useful biological properties.

14 cl, 1 dwg, 12 tbl, 19 ex

 

The text descriptions are given in facsimile form.

1. Expression vector for production in E. coli protein IL-21, containing the following functionally connected elements:
(a) a replication origin;
(b) a DNA element of transcription initiation;
(c) the polynucleotide sequence presented as SEQ ID NO:27, and
(d) a transcription terminator.

2. The expression vector according to claim 1, which further comprises breeding the token.

3. Expression vector rtar for production in E. coli the protein encoded by the nucleotide sequence presented as SEQ ID NO:27, which are deposited in ATSC number of MOUTH-4853.

4. A host cell E. coli transformed with the expression vector according to any one of claims 1 to 3.

5. A host cell according to claim 4, which is a cell strain E. coli W3110.

6. The method of producing the protein of IL-21, including:
(a) to livelounge host cells according to claim 5 in environment, suitable for the expression of IL-21;
(b) removing cells from the environment for cultivation;
(c) isolation of cell protein IL-21.

7. The method of producing the protein of IL-21, including:
(a) culturing host cells according to claim 5 in environment for growing periodic fermentation injection;
(b) removing cells from the environment for cultivation;
(c) isolation of cell protein IL-21.

8. The method of producing the protein of IL-21, including:
(a) culturing host cells according to claim 4 or 5 in shake flask until OD600 of 5 to 20 in the medium for cultivation;
(b) seeding apparatus for fermentation from 1 to 12% vol./about. the contents of the shake flask;
(c) culturing the cells in the apparatus in the growing medium at pH from 6.2 to 7.2, in which nutrient solution previously served 15 h actual duration of fermentation (EFT);
(d) adding 20-30 hours actual duration of fermentation inducing agent;
(e) collection of host cells in the range from 48 to 56 hours of actual duration of fermentation;
(f) isolation of cell protein IL-21.

9. The method according to claim 8, in which the inducing agent is isopropylcyclopentadienyl (IPTG) at the rate of 0.5 to 2 mm.

10. The method of claim 8, where the nutrient solution contains a carbohydrate selected from the group consisting of glycerol and glucose at a concentration of media for growing and velocity Pete the Oia 5-15 grams of carbohydrate per hour.

11. The method according to claim 10, where the glycerol is 40-70% (vol./about.) glycerol and glucose is 40-70% (wt./about.) glucose.

12. The method according to claim 10, where the content of glycerin is about 70% (vol./about.) or the glucose content is about 60% (wt./vol.).

13. The method of producing the protein of IL-21, including:
(a) preparation of seed flasks with inoculum containing cell host according to claim 5, and with culture medium containing approximately 5 g/l glycerol;
(b) culturing the inoculum in the environment for growing for 16-20 h at approximately 30C;
(c) the transfer of the cultured supernatant in the culture medium in the fermentor for periodic cultivation in a concentration of 0.5-5% (vol./vol.);
(d) fermentation periodic cultivation at about 37C. and pH of about 6.8 in the presence of approximately 2% glycerol;
(e) commencement of injection at 8 o'clock the actual duration of the fermentation of glucose supply with parameters corresponding to about 9.5 g/l/h, and continuing this procedure until the expiration time of fermentation;
(f) adding at the 24 hour IPTG to a final concentration of from 0.5 to 2 mm;
(g) the fermentation is approximately 28 h after addition of IPTG;
(h) discharge of fermentation medium from the fermenter;
(i) adding to the fermentation medium is approximately equal volume of water;
(j) homogenization and centrifugation, the enzyme is operating environment for the collection of sediment or suspension cells, containing protein material;
(k) the selection of cell protein IL-21.

14. A host cell strain, designated ZGOLD1 and deposited in ATSC number of MOUTH - 5698, which transformed vector rtar described in claim 3, producing IL-21.
Priority items:

13.12.2002 according to claims 1-14.



 

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