Production and purification of il-29

Abstract

The expression vectors and methods using an E. Coli expression system for the large scale production of IL-29 are described. The vectors utilize the IL-29 coding sequence with specific changes in nucleotides in order to optimize codons and mRNA secondary structure for translation in E. coli . Also included are methods of producing, purifying and pegylating an IL-29 polypeptide.

1 . A method of producing an IL-29 polypeptide comprising:
 (a) culturing a prokaryotic host cell comprising a nucleic acid molecule encoding an IL-29 polypeptide operably linked to an inducible promoter in a first growth medium under conditions wherein the encoded IL-29 polypeptide is expressed in a shake flask to an OD600 of 5 to 20;   (b) inoculating a fermentation vessel with 1 to 5% v/v of shake flask medium containing host cells;   (c) culturing the host cells in a second growth medium at a pH of 6.2 to 7.2, wherein a carbohydrate feed solution is fed into the fermentation vessel at 6 to 8 hours elapsed fermentation time;   (d) adding an inducing agent to the fermentation vessel at 20 to 30 hours elapsed fermentation time; and   (e) harvesting the prokaryotic host cells at 48 to 56 hours elapsed fermentation time.   
     
     
         2 . The method of  claim 1 , wherein the carbohydrate feed solution comprises a glycerol or glucose at a concentration of 10 to 30 g/L growth medium, and a feed rate of 5-15 grams of glycerol or glucose per liter per hour. 
     
     
         3 . The method of  claim 1  wherein the prokaryotic host cell is  Escherichia coli.    
     
     
         4 . The method of  claim 3  wherein the  Escherichia coli  cell is W3110. 
     
     
         5 . The method of  claim 3  wherein the  Escherichia coli  cell is ZGOLD1. 
     
     
         6 . The method of  claim 3  wherein the  Escherichia coli  cell is OmpT deficient. 
     
     
         7 . The method of  claim 3  wherein the  Escherichia coli  cell is ZGOLD5. 
     
     
         8 . The method of  claim 3  wherein the  Escherichia coli  cell is fhuA deficient. 
     
     
         9 . The method of  claim 3  wherein the  Escherichia coli  cell is OmpT and fhuA deficient. 
     
     
         10 . The method of  claim 1  wherein the encoded IL-29 polypeptide is selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10 and 12. 
     
     
         11 . The method of  claim 1  wherein the inducing agent of step (d) is isopropyl thiogalactopyranoside. 
     
     
         12 . The method of  claim 11  wherein isopropyl thiogalactopyranoside is added to the culture at a concentration of 0.5 mM to 2 mM. 
     
     
         13 . A method of recovering an IL-29 polypeptide from a prokaryotic host cell comprising:
 (a) culturing a prokaryotic host cell comprising a nucleic acid molecule encoding an IL-29 polypeptide operably linked to an inducible promoter in growth medium under conditions wherein the encoded IL-29 polypeptide is expressed;   (b) adding an inducing agent to induce expression of the IL-29 polypeptide;   (c) harvesting the prokaryotic host cells;   (d) lysing the prokaryotic host cells;   (e) centrifuging the lysed prokaryotic host cells;   (f) recovering the inclusion body pellet;   (g) solubilizing the inclusion body pellet in 4-6 M guanidine hydrochloride and 10-50 mM dithiothreitol for 1-2 hours at 15-25° C.; and   (h) adding the solubilized IL-29 polypeptide to a refolding buffer comprising 0.05-0.5% polyethylene glycol, salt, 0.5 M-1.25 M arginine and a mixture of reduced and oxidized molecules for 1-26 hours at a temperature of 4-30° C. and a pH 7.3-8.5, wherein the solubilized IL-29 polypeptide is refolded;   (i) quenching the refolding reaction by adjusting the pH to 5.5-6.5;   (j) diluting the quenched refolding solution 1.5- to 10-fold in water or low ionic strength buffer at pH 5-7; and   (k) filtering the quenched, diluted refold solution through filters to remove precipitate or particulates.   
     
     
         14 . The method of  claim 13  wherein the prokaryotic host cells of step (d) are lysed by homogenization. 
     
     
         15 . The method of  claim 13  wherein the lysed prokaryotic host cells of step (e) are centifuged by either batch or continuous centrifugation. 
     
     
         16 . The method of  claim 13  wherein the IL-29 polypeptide of step (h) is added to the refolding buffer to a final concentration of 0.05-3.0 mg/ml. 
     
     
         17 . The method of  claim 13  wherein the mixture of reduced and oxidized molecules of the refolding buffer are selected from the group consisting of cysteine and cystine, dithiothreitol and cystine, reduced glutathione and oxidized glutathione, and dithiothreitol and oxidized glutathione. 
     
     
         18 . A method of purifying an IL-29 polypeptide comprising:
 (a) providing the IL-29 polypeptide according to step (k) of  claim 13 ;   (b) loading the filtered solution comprising refolded IL-29 polypeptide of step (a) onto a cation exchange chromatography column equilibrated with sodium acetate at pH 5.5;   (c) eluting bound IL-29 polypeptide with sodium chloride in sodium acetate, pH 5.5; and   (d) adjusting the eluate with ammonium sulfate to 1 M concentration, and passing the adjusted IL-29 polypeptide eluate through a 0.45 μm filter.   
     
     
         19 . The method of  claim 18  wherein the IL-29 polypeptide elutes from the cation exchange column to form a pool at about 0.7 M-0.8 M sodium chloride after using linear gradient elution of 0-2M sodium chloride. 
     
     
         20 . The method of  claim 18  further comprising:
 (e) loading the IL-29 polypeptide of step (d) onto a hydrophobic interaction chromatography column equilibrated with 50 mM sodium acetate, 1.5 M ammonium sulfate, pH 5.5;   (f) eluting the IL-29 polypeptide with a linear 50 mM sodium acetate, 1.5 M ammonium sulfate to 50 mM sodium acetate with no ammonium sulfate, pH 5.5;   (g) diluting the eluate about 6-fold with water or low ionic strength buffer and passing the diluted IL-29 polypeptide eluate through a 0.2 μm or 0.45 μm filter.   
     
     
         21 . The method of  claim 20  wherein the IL-29 polypeptide elutes from the hydrophobic interaction chromatography column at about 0.75 M ammonium sulfate to 0 M ammonium sulfate. 
     
     
         22 . The method of  claim 20  further comprising:
 (h) loading the IL-29 polypeptide of step (g) onto a high performance cation exchange chromatography column equilibrated with 50 mM sodium acetate comprising 0-300 mM sodium chloride, pH 5.5; and   (i) eluting the IL-29 polypeptide with a higher concentration of sodium chloride in 50 mM sodium acetate, pH 5.5, in a step or gradient elution format.   
     
     
         23 . The method of  claim 22  wherein the IL-29 polypeptide elutes from the high performance cation exchange chromatography column at about 0.4 M sodium chloride to 0.6 M sodium chloride after using a gradient elution of 300 to 800 mM sodium chloride. 
     
     
         24 . A method of concentrating IL-29 polypeptide comprising:
 (a) providing the IL-29 polypeptide according to step (i) of  claim 22 ;   (b) adding the IL-29 polypeptide to a tangential flow filtration plate and frame system comprising one or more a 3-10 kDa molecular weight cut-off membrane;   (c) applying a transmembrane pressure of 15-25 psi to the system to ultrafilter the solution to a higher concentration; and   (c) filtering the concentrated IL-29 polypeptide through a 0.2 μm membrane.   
     
     
         25 . A method of monopegylating an IL-29 polypeptide comprising:
 (a) providing 3-5 g/L IL-29 polypeptide in a sodium acetate buffer solution;   (b) adding 10-20 mM sodium cyanoborohydride to the solution of step   (a);   (c) adding a 2-fold molar excess of derivatized polyethylene glycol to the solution of step (b); and   (d) mixing the solution of step (c) for 10-18 hours at 16-20° C.   
     
     
         26 . A method of purifying monopegylated IL-29 polypeptide comprising:
 (e) providing the monopegylated IL-29 polypeptide according to step (d) of  claim 25 ;   (f) diluting the solution of step (e) 2-fold with 50 mM sodium acetate, pH 5.5;   (g) filtering the solution of step (f) through a 0.2 μm membrane;   (h) loading the solution of step (g) onto a high performance cation exchange chromatography column equilibrated with 50 mM sodium acetate, 200 mM sodium chloride, pH 5.5;   (i) eluting monopegylated IL-29 polypeptide from the high performance cation exchange chromatography column with a linear 50 mM sodium acetate, 500 mM sodium chloride gradient, pH 5.5;   (j) adding the monopegylated IL-29 polypeptide to a tangential flow filtration plate and frame system comprising one or more a 3-10 kDa molecular weight cut-off membrane;   (k) applying a transmembrane pressure of 15-25 psi to the system to ultrafilter the solution to a higher concentration;   (l) using the system to buffer exchange the concentrated IL-29 polypeptide into an appropriate formulation buffer by diafiltration; and   (m) filtering the concentrated monopegylated IL-29 polypeptide through a 0.2 μm membrane.   
     
     
         27 . The method of  claim 26  wherein the polyethylene glycol comprises 20 kDa or 30 kDa mono-methoxyPEG-propionaldehyde. 
     
     
         28 . The method of  claim 26  wherein the polyethylene glycol is N-terminally attached to the IL-29 polypeptide. 
     
     
         29 . The method of  claim 22  wherein the IL-29 polypeptide is at least 98% pure by sodium dodecyl sulfate polyacrylamide gel analysis and aggregates are less than 0.2% by size exclusion HPLC. 
     
     
         30 . The method of  claim 24  wherein the IL-29 polypeptide is at least 98% pure by sodium dodecyl sulfate polyacrylamide gel analysis and aggregates are less than 0.2% by size exclusion HPLC. 
     
     
         31 . The method of  claim 24  wherein the IL-29 polypeptide has an endotoxin level of less than 10 endotoxin units per milligram of IL-29 polypeptide in a  Limulus  amoebocyte lysate assay based on USP <85>. 
     
     
         32 . The method of  claim 25  wherein the monopegylated IL-29 polypeptide is at least 99% monopegylated as measured by reversed phase HPLC. 
     
     
         33 . An IL-29 polypeptide produced by the method of  claim 1 . 
     
     
         34 . An IL-29 polypeptide produced by the method of  claim 22 . 
     
     
         35 . An IL-29 polypeptide produced by the method of  claim 24 . 
     
     
         36 . A monopegylated IL-29 polypeptide produced by the method of  claim 25 . 
     
     
         37 . A purified monopegylated IL-29 polypeptide produced by the method of  claim 26 .