# US 7,745,176 B2 · IL-21 production in prokaryotic hosts > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,745,176 B2 - Document ID: US7745176B2 - Status: Granted - Kind: B2 - Application number: 11/539,036 - Filed: 2006-10-05 - Published: 2007-03-01 - Granted: 2010-06-29 - Earliest claimed: 2002-12-13 - Assignees: ZymoGenetics, Inc. - Inventors: Bruce L. Zamost; Douglas C. Covert - Web page: https://patentgrep.com/patent/US7745176B2 ## CPC - C07K 14/54: Interleukins [IL] - 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 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. ## Claims ### Claim 1 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. ### Claim 2 (depends on 1) The method of claim 1, wherein the step (a) further comprises: (1) culturing the host cell in a shake flask to an OD600 of 5 to 20 in a growth medium; (2) inoculating a fermentation vessel with the shake flask medium containing the host cell; (3) culturing the host cell in a growth medium at a pH of 6.2 to 7.2, wherein a feed solution is fed into the fermentation vessel before 15 hours elapsed fermentation time (EFT); (4) adding an inducing agent to the fermentation vessel at 20 m 30 hours EFT; and (5) harvesting the host cell at 48 to 56 hours EFT. ### Claim 3 (depends on 2) The method of claim 2, wherein the host cell is E. coli strain W3110. ### Claim 4 (depends on 2) The method of claim 2, wherein the inducing agent is isopropyl thiogalactopyranoside (IPTG) at 0.5 to 2 mM. ### Claim 5 (depends on 2) The method of claim 2, wherein the feed solution comprises a carbohydrate selected from the group consisting of glycerol and glucose at a feed rate of 5-15 grams of carbohydrate per hour. ### Claim 6 (depends on 5) The method of claim 5, wherein the glycerol is at a concentration of 40 to 70% v/v or the glucose is at a concentration of 40 to 70% w/v. ### Claim 7 (depends on 5) The method of claim 5, wherein the glycerol is at a concentration of about 70% v/v or the glucose is at a concentration of about 60% w/v. ### Claim 8 (depends on 5) The method of claim 5, wherein the fermentation vessel is inoculated with 1 to 12% v/v of the growth medium containing the host cells. ### Claim 9 (depends on 2) The method of claim 2, wherein the feed solution comprises yeast extract. ### Claim 10 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. ### Claim 11 (depends on 10) The method according to claim 10, wherein the biological activity of the IL-21 protein is measured using an IL-21 receptor-binding cell assay.