IL-21 production in prokaryotic hosts
AssigneeZymoGenetics, Inc.
InventorsBruce L. Zamost, Douglas C. Covert
Life of this patent
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Abstract
The expression vectors and methods using an E. coli expression system for the large scale production of IL-21 are described. The vectors utilize the IL-21 coding sequence with specific changes in nucleotides in order to optimize codons and mRNA secondary structure for translation in E. coli. Using the expression vectors, the IL-21 gene was produced in E. coli to a level of greater than 1 g/L in fed batch fermentation. Also included are OmpT deficient E. coli strains transformed with an IL-21 expression vector.
Claims11
A method for producing an IL-21 protein comprising:
- (a) culturing a host cell in growth medium by fed batch fermentation, wherein the host cell is transformed with an expression vector for producing the IL-21 protein comprising the following operably linked elements:
- (i) a prokaryotic origin of replication;
- (ii) a transcriptional initiation DNA element;
- (iii) a polynucleotide sequence comprising SEQ ID NO:27;
- and
- (iv) a transcriptional terminator;
- (b) recovering the host cell from the growth medium;
- and
- (c) isolating the IL-21 protein from the host cell.
A method of producing an IL-21 protein comprising:
- (a) seeding a flask with an inoculum comprising an E. coli W3110 host cell expressing an IL-21 polypeptide comprising SEQ ID NO:28, or an E. coli W3110 host cell comprising pTAP337 vector wherein the IL-21 polypeptide is expressed, and with growth medium comprising about 5 g/L glycerol;
- (b) culturing the inoculum in growth medium for 16-20 hours at about 30° C.;
- (c) transferring the cultured inoculum in growth medium to a batch fermentor at a concentration of 0.5-5% v/v inoculum;
- (d) fermenting the batch fermentation at about 37° and about pH 6.8;
- with about 2% glycerol;
- (e) introducing a glucose feed at about 8 hours elapsed fermentation time (EFT) of about 9.5 g glucose/liter/hour and continuing until end of a fermentation run;
- (f) adding IPTG at about 24 hour EFT to final concentration of 0.5 to 2 mM;
- (g) fermenting about 28 hours after addition of IPTG;
- (h) harvesting fermentation broth from the fermenter;
- (i) adding an equal volume of water to the fermentation broth;
- (j) homogenizing and centrifuging the fermentation broth to collect a cell pellet or cell slurry comprising IL-21 protein material;
- and
- (k) isolation of the IL-21 protein from the cell pellet or cell slurry.
Dependencies are inferred from the claim text.