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US 7,622,561 B2Granted
IL-21 production in prokaryotic hosts
AssigneeZymoGenetics, Inc.
InventorsBruce L. Zamost, Hong Y. Liu, Karen S. De Jongh
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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.
Claims8
A method for isolating an IL-21 protein comprising SEQ ID NO:28 comprising the steps of:
- (a) separating host cells expressing the IL-21 protein from a growth medium;
- (b) disrupting the host cells to collect a cell pellet or cell slurry;
- (c) separating water insoluble IL-21 protein material from the cell pellet or cell slurry;
- (d) dissolving the insoluble IL-21 protein material in a chaotropic solvent;
- (e) diluting the chaotropic solvent and refolding the IL-21 protein;
- and
- (f) isolating the IL-21 protein, wherein the isolated IL-21 protein is biologically active.
4 more elements
A method for isolating an IL-21 protein comprising SEQ ID NO:28 comprising the steps of:
- (a) separating from a fermentation broth host cells expressing the IL-21 protein;
- (b) disrupting the host cells to collect a cell pellet or cell slurry comprising water insoluble IL-21 protein material;
- (c) homogenizing the cell pellet or cell slurry to collect inclusion bodies;
- (d) dissolving the insoluble IL-21 protein material of the inclusion bodies in a chaotropic solvent comprising a guanidine salt;
- (e) diluting the chaotropic solvent by addition of a refolding buffer comprising arginine salts and a mixture of reducing and oxiding components;
- (f) isolating the IL-21 protein by removing unfolded and aggregated proteins by filtering;
- and
- (g) purifying the IL-21 refolded protein on a cation exchange column;
- wherein the isolated and purified IL-21 protein is biologically active.
6 more elements
A method for isolating an IL-21 protein comprising SEQ ID NO:28 comprising the steps of:
- (a) separating from a fermentation broth host cells expressing the IL-21 protein;
- (b) disrupting the host cells to collect a cell pellet or cell slurry comprising water insoluble IL-21 protein material;
- (c) homogenizing the cell pellet or cell slurry to collect inclusion bodies;
- (d) dissolving the insoluble IL-21 protein material of the inclusion bodies in a chaotropic solvent comprising a guanidine salt;
- and
- (e) diluting the chaotropic solvent by addition of a refolding buffer comprising arginine salts and a mixture of reducing and oxidizing components;
- (f) isolating the IL-21 protein by removing unfolded and aggregated proteins by filtering;
- (g) purifying the IL-21 refolded protein on a cation exchange column;
- and
- (h) purifying an IL-21 eluate resulting from step (g) on a hydrophobic interaction column, wherein the isolated and purified IL-21 protein is biologically active.
7 more elements
A method for isolating an IL-21 protein comprising SEQ ID NO:28 comprising the steps of:
- (a) separating from a fermentation broth host cells expressing the IL-21 protein;
- (b) disrupting the host cell to collect a cell pellet or cell slurry comprising water insoluble IL-21 protein material;
- (c) homogenizing the cell pellet or cell slurry to collect inclusion bodies;
- (d) dissolving the insoluble IL-21 protein of the inclusion bodies in a chaotropic solvent comprising about 6M guanidine hydrochloride, 40 mM dithiothreitol (DTT) for about one hour at room temperature;
- (e) refolding the dissolved IL-21 protein 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;
- (f) adjusting pH to about 5.5 with about 20% acetic acid and allowing the solution to react for at least five hours;
- (g) diluting the solution with about 1+1.4 volumes 25 mM acetate, pH 5.5;
- (h) filtering the solution;
- (i) loading the solution on a resin column equilibrated to pH 5.5 using sodium acetate buffer;
- (j) washing the resin column with about 0.4 M sodium chloride;
- (k) washing the resin column with about 0.75 M sodium chloride to elute bound IL-21 protein;
- (l) adding ammonium sulfate to a concentration of about 1.5 M to the eluate and filtering the eluate solution;
- (m) loading the eluate solution onto a Tosohaas butyl 650-M column equilibrated to 1.5 M ammonium sulfate, 0.05 M sodium chloride in sodium acetate buffer;
- (n) washing the column with about 0.15 M ammonium sulfate, 0.05 sodium chloride in sodium acetate buffer;
- (o) diluting the eluate solution to a conductivity of about 30 mS/cm with water;
- (p) loading the eluate solution onto a SP Sepharose HP column equilibrated with sodium acetate buffer;
- (q) washing the column with 20-column volume linear gradient from 0.3 to 0.7 M sodium chloride;
- (r) concentrating the IL-21 protein;
- and
- (s) exchanging buffer to formulation buffer using tangential flow ultrafiltration.
17 more elements
Dependencies are inferred from the claim text.