IL-21 production in prokaryotic hosts
AssigneeZymoGenetics, Inc.
InventorsChung Chan, Bruce Zamost, Douglas Covert, Hong Liu, Karen De Jongh, Jeffrey Meyer, Susan Holderman
Life of this patent
Estimating the term…
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.
Claims22
An expression vector for producing IL-21 protein comprising the following operably linked elements:
- (a) a prokaryotic origin of replication;
- (b) a transcriptional initiation DNA element;
- (c) a polynucleotide sequence as shown in SEQ ID NO:27;
- and
- (d) a transcriptional terminator.
An expression vector comprising the pTAP337 vector, deposited with the American Type Culture Collection in Manassas, Va. under Patent Deposit Designation PTA-4853.
A method of producing IL-21 protein comprising:
- (a) seeding a flask with an inoculum comprising an E. coli W3110 host cell expressing an IL-21 polypeptide as shown in SEQ ID NO:28, or an E. coli W3110 host cell comprising pTAP337 vector wherein an 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 EFF 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;
- and
- (0) homogenizing and centrifuging the fermentation broth to collect a cell pellet or cell slurry comprising IL-21 protein material.
A method for isolating insoluble IL-21 protein comprising a sequence of amino acid residues as shown in SEQ ID NO:28 comprising the steps of:
- (a) separating water insoluble IL-21 protein material from a cell pellet or cell slurry;
- (b) dissolving the insoluble IL-21 protein material in a chaotropic solvent;
- (c) diluting the chaotropic solvent and refolding the IL-21 protein;
- and
- (d) isolating the IL-21 protein, wherein the isolated IL-21 protein is capable of being biologically active.
A method for isolating insoluble IL-21 protein comprising a sequence of amino acid residues as shown in SEQ ID NO:28 comprising the steps of:
- (a) separating from a fermentation broth a cell pellet or cell slurry comprising water insoluble IL-21 protein material;
- (b) homogenizing the cell pellet or cell slurry to collect inclusion bodies;
- (c) dissolving the insoluble IL-21 protein material in a chaotropic solvent comprising a guanidine salt;
- (d) diluting the chaotropic solvent by addition of a refolding buffer comprising arginine salts and a mixture of reducing and oxiding components;
- (e) isolating the IL-21 protein by removing unfolded and aggregated proteins by filtering;
- and
- (f) purifying the IL-21 refolded protein on a cation exchange column;
- wherein the isolated and purified IL-21 protein is capable of being biologically active.
A method for isolating insoluble IL-21 protein comprising a sequence of amino acid residues as shown in SEQ ID NO:28 comprising the steps of:
- (a) separating from a fermentation broth a cell pellet or cell slurry comprising water insoluble IL-21 protein material;
- (b) homogenizing the cell pellet or cell slurry to collect inclusion bodies;
- (c) dissolving the insoluble IL-21 protein material in a chaotropic solvent comprising a guanidine salt;
- and
- (d) diluting the chaotropic solvent by addition of a refolding buffer comprising arginine salts and a mixture of reducing and oxidizing components;
- (e) isolating the IL-21 protein by removing unfolded and aggregated proteins by filtering;
- (f) purifying the IL-21 refolded protein on a cation exchange column;
- and
- (g) purifying the IL-21 eluate from step (f) on a hydrophobic interaction column, wherein the isolated and purified IL-21 protein is capable of being biologically active.
A method for isolating insoluble IL-21 protein comprising a sequence of amino acid residues as shown in SEQ ID NO:28 comprising the steps of:
- (a) separating from a fermentation broth a cell pellet or cell slurry comprising water insoluble IL-21 protein material;
- (b) homogenizing the cell pellet or cell slurry to collect inclusion bodies;
- (c) dissolving the insoluble IL-21 protein in a chaotropic solvent comprising about 6M guanidine hydrochloride, 40 mM dithiothreitol (DTT) for about one hour at room temperature;
- (d) refolding the dissolved inclusion bodies in a solution by diluting into refolding buffer comprising about 0.75 M arginine, 2 mM DTT/4 mM cystine oxidation-reduction pair at least 20 times;
- (e) adjusting pH to about 5.5 with about 20% acetic and allowing the solution to react for at least five hours;
- (f) diluting the solution with about 1+1.4 volumes 25 mM acetate, pH 5.5;
- (g) filtering the solution;
- (h) loading solution on resin column equilibrated to pH 5.5 using sodium acetate buffer;
- (i) washing the resin column with about 0.4 M sodium chloride;
- (j) washing the resin column with about 0.75 M sodium chloride to elute bound IL-21 protein;
- (k) adding ammonium sulfate to a concentration of about 1.5 M to eluate and filtering eluate solution;
- (1) loading eluate onto a Tosohaas butyl 650-M column equilibrated to 1.5 M ammonium sulfate, 0.05 M sodium chloride in sodium acetate buffer;
- (m) washing column with about 0.15 M ammonium sulfate, 0.05 sodium chloride in sodium acetate buffer;
- (n) diluting the eluate to a conductivity of about 30 mS/cm with water;
- (o) loading eluate onto a SP Sepharose HP column equilibrated with sodium acetate buffer;
- (p) washing column with 20-column volume linear gradient from 0.3 to 0.7 M sodium chloride;
- (q) concentrating the IL-21 protein;
- and
- (r) exchanging buffer to formulation buffer using tangential flow ultrafiltration.
A composition comprising an IL-21 protein comprising amino acids residues 1-163 as shown in SEQ ID NO:28 at a concentration of about 10 mg/ml IL-21 protein in about 10 mM histidine, 4.7% mannitol at pH 5.3
A host cell from a strain of zGOLD1, deposited with the American Type Culture Collection (ATCC) in Manassas, Va. transformed with an expression vector comprising a pTAP337 vector, deposited with the ATCC under Patent Deposit Designation PTA-4853.
Dependencies are inferred from the claim text.