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1. (WO1991006634) IMPROVED METHOD FOR PURIFICATION OF RECOMBINANT COPPER/ZINC (CU-ZN) SUPEROXIDE DISMUTASE FROM BACTERIA OR EUCARYOTIC CELLS
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What is claimed is:

1. A method Sor recovering a solution containing purified, enzymatically active Cu-Zn superoxide dismutase or a polypeptide analog thereof having substantially the same amino acid sequence as, and the biological activity of, naturally-occurring Cu-Zn superoxide dismutase from a composition which comprises cells containing Cu-Zn superoxide dismutase or a polypeptide analog thereof comprising:

(a) treating the composition so as to separate soluble proteins present in the cells from whole cells, cellular debris and insoluble proteins so as to obtain a solution containing such soluble proteins;

(b) treating the resulting solution containing the soluble proteins with a second solution containing a salt at a concentration such that the soluble proteins other than
Cu-Zn superoxide dismutase or the polypeptide analog thereof present in the solution containing the soluble proteins are rendered capable of binding to an appropriate hydrophobic substance;

(c) contacting the then-resulting solution containing the soluble proteins with an appropriate hydrophobic substance so as to bind the soluble proteins other than Cu-Zn superoxide dismutase or the polypeptide analog thereof present in such solution to the hydrophobic substance and thus separate such other proteins from the Cu-Zn superoxide dismutase or polypeptide analog thereof; and

(d) recovering the resulting solution containing purified, enzymatically active Cu-Zn superoxide dismutase or polypeptide analog thereof.

2. A method of claim 1, wherein the Cu-Zn superoxide dismutase or polypeptide analog thereof is human Cu-Zn superoxide dismutase or a polypeptide analog thereof.

3. A method of claim 2, wherein the polypeptide analog comprises a non-acetylated polypeptide analog of human Cu-Zn superoxide dismutase.

4. A method of claim 1, wherein the cells comprise bacterial cells.

5. A method of claim 4, wherein the bacterial cells comprise cells of an Escherichia coli strain.

6. A method of claim 5, wherein the Escherichia coli strain comprises Escherichia coli A4255 containing the plasmid designated pSODβ1T11 and deposited under ATCC Accession No. 53468.

7. A method of claim 5, wherein the Escherichia coli strain comprises Escherichia coli A4255 containing the plasmid pSODβMAX-12 and deposited under ATCC Accession No. 67177.

8. A method of claim 5, wherein the Escherichia coli strain comprises Escherichia coli A2097 containing the plasmid designated pSODα2 and deposited under ATCC Accession No. 39786.

9. A method of claim 5, wherein the Escherichia coli strain comprises Escherichia coli SΦ732 containing the plasmid designated pMF2005 and deposited under ATCC Accession No. 67362.

10. A method of claim 5, wherein the Escherichia coli strain comprises Escherichia coli SΦ930 containing the plasmid designated pMF5534 and deposited under ATCC Accession No. 67703.

11. A method of claim 5, wherein the Escherichia coli strain comprises Escherichia coli A1645 containing the plasmid designated pNd-S0DNN-12 and deposited under ATCC Accession No. 53166.

12. A method of claim 1, wherein the cells comprise eucaryotic cells.

13. A method of claim 12, wherein the eucaryotic cells comprise yeast cells.

14. A method of claim 12, wherein the eucaryotic cells comprise mammalian cells.

15. A method of claim 1, wherein the treatment of the composition in step (a) comprises treating the composition so as to disrupt the cells and obtain a cellular extract therefrom, and then subjecting the resulting cellular extract to centrifugation so as to obtain the solution containing the soluble proteins.

16. A method of claim 1, wherein the salt in the solution of step (b) is selected from the group consisting of sodium sulfate, sodium chloride, ammonium sulfate, potassium chloride, ammonium acetate or a combination of any of the foregoing.

17. A method of claim 1, wherein the appropriate hydrophobic substance comprises phenyl-Sepharose.

18. A method of claim 1, wherein the appropriate hydrophobic substance comprises a suitable resin having a phenyl functional group.

19. A method of claim 18, wherein the suitable resin is a polysaccharide resin having phenyl groups present thereon.

20. A method of claim 1, wherein the contacting of step (c) is effected by passing the solution containing the soluble proteins through a column containing the hydrophobic substance.

21. A method of claim 1, which further comprises treating the resulting solution containing the soluble proteins from step (a) so as to obtain a more concentrated solution containing purified Cu- Zn superoxide dismutase or polypeptide analog thereof.

22. A method of claim 21, wherein the treatment of the solution to obtain a more concentrated solution of purified Cu-Zn superoxide dismutase or polypeptide analog thereof comprises ultrafiltration.

23. A method of claim 22, wherein the resulting more concentrated solution of purified Cu-Zn superoxide dismutase or polypeptide analog thereof comprises a, b and c isoforms of Cu-Zn superoxide dismutase or polypeptide analog thereof.

24. A method of claim 23, which further comprises treating the resulting more concentrated solution of purified Cu-Zn superoxide dismutase or polypeptide analog thereof so as to produce three separate solutions, each of which has an increased concentration of one of either of the a, b or c isoform.

25. A method of claim 24, wherein the treatment of the concentrated solution comprises anion exchange chromatography.

26. A method of claim 25, which further comprises subjecting the then-resulting solution which has an increased concentration of the b isoform of purified Cu-Zn superoxide dismutase or polypeptide analog thereof to cation exchange chromatography so as to produce three further separate solutions.

each of which has a further increased concentration of one of either of the a, b or c isoform.

27. A method of claim 26, which further comprises treating the then-resulting solution which has a further increased concentration of the b isoform of purified Cu-Zn superoxide dismutase or polypeptide analog thereof so as to further purify the Cu-Zn superoxide dismutase or polypeptide analog thereof contained therein prior to treating the resulting solution containing the further purified Cu-Zn superoxide dismutase or polypeptide analog thereof with a second solution containing a salt in step (b).

28. A method of claim 27, wherein the treatment of the then-resulting solution comprises strong anion exchange chromatography.

29. A method of claim 28, wherein the strong anion exchange chromatography comprises passing the then-resulting solution through a column of a polysaccharide resin having quaternary amino function groups present thereon.

30. A method of claim 25, which further comprises treating the then-resulting solutions which have an increased concentration of one of either of the a or c isoform so as to increase the concentration of b isoform and reduce the concentration of a isoform and c isoform of the polypeptide analog contained in each of the solutions prior to treating the then-resulting solution containing the further purified Cu-Zn superoxide dismutase or polypeptide analog thereof with a second solution containing a salt in step (b).

31. A method of claim 26, which further comprises treating the then-resulting solutions which have a further increased concentration of one of either of the a or c isoform so as to increase the concentration of b isoform and reduce the concentration of a isoform and c isoform of the polypeptide analog contained in each of the solutions prior to treating the then-resulting solution containing the further purified Cu-Zn superoxide dismutase or polypeptide analog thereof with a second solution containing a salt in step (b).

32. A method of claim 30 or 31, wherein the treatment of the then-resulting solutions prior to step (b) comprises:

(a) combining the separate solution which has an increased concentration of the a isoform with the separate solution which has an increased concentration of the c isoform;

(b) treating the resulting combined solution so as to produce a solution which has an increased concentration of the b isoform;

(c) recovering the then-resulting solution which has an increased concentration of the b isoform.

33. A method of claim 32, wherein the treatment of the resulting combined solution in step (b) comprises incubation.

34. A method of claim 32, which further comprises treating the solution with an increased concentration of the b isoform from step (b) so as to purify the b isoform present in the solution.

35. A method of claim 34, wherein the treatment of the solution comprises ion exchange chromatography.

36. A method of claim 35, wherein the ion exchange chromatography comprises anion exchange chromatography.

37. A method of claim 36, wherein the ion exchange chromatography further comprises cation exchange chromatography.

38. A method of claim 1, which further comprises treating the resulting solution containing purified, enzymatically active Cu-Zn superoxide dismutase or polypeptide analog thereof recovered in step (d) so as to obtain a more concentrated solution containing purified Cu-Zn superoxide dismutase or polypeptide analog thereof.

39. A method of claim 38, wherein the treatment of the resulting solution to obtain a more concentrated solution of purified Cu-Zn superoxide dismutase or polypeptide analog thereof comprises ultra-filtration.

40. A method of claim 38, which further comprises treating the resulting more concentrated solution containing purified Cu-Zn superoxide dismutase or polypeptide analog thereof so as to obtain a solution containing more purified Cu-Zn superoxide dismutase or polypeptide analog thereof.

41. A method of claim 40, wherein the treatment of the resulting solution comprises strong anion exchange chromatography.

42. A method of claim 41, wherein the strong anion exchange chromatography comprises passing the resulting solution through a column of a polysaccharide resin having quaternary amino functional groups present thereon.

43. A method of claim 1, which further comprises treating the resulting solution containing purified, enzymatically active Cu-Zn superoxide dismutase or polypeptide analog thereof recovered in step (d) so as to obtain a solution containing more purified Cu-Zn superoxide dismutase or polypeptide analog thereof.

44. A method of claim 43, wherein the treatment of the resulting solution comprises strong anion exchange chromatography.

45. A method of claim 44, wherein the strong anion exchange chromatography comprises passing the resulting solution through a column of a polysaccharide resin having quaternary amino functional groups present thereon.

46. A method of claim 1, which further comprises treating the resulting solution containing the soluble proteins from step (a) so as to purify the Cu-Zn superoxide dismutase or polypeptide analog thereof contained therein prior to treating the resulting solution containing the soluble proteins with a second solution containing a salt in step (b).

47. A method of claim 46, wherein the treatment of the resulting solution comprises strong anion exchange chromatography.

48. A method of claim 47, wherein the strong anion exchange chromatography comprises passing the resulting solution through a column of a polysaccharide resin having quaternary amino functional groups present thereon.

49. A method of claim 1, which further comprises treating the resulting solution containing the soluble proteins from step (a), the soluble proteins including Cu-Zn superoxide dismutase or polypeptide analog thereof comprising a, b and c isoforms of Cu-Zn superoxide dismutase or polypeptide analog thereof, so as to increase the concentration of b isoform and reduce the concentration of a isoform and c isoform of the polypeptide analog contained in the solution prior to treating the resulting solution containing the soluble proteins with a second solution containing a salt in step (b).

50. A method of claim 49, wherein the treatment of the resulting solution prior to step (b) comprises:

(a) treating the resulting solution containing the soluble proteins so as to produce three separate solutions, each of which has an increased concentration of one of either of the a, b, or c isoform;

(b) recovering the separate solution which has an increased concentration of the b isoform;

(c) combining the separate solution which has an increased concentration of the a isoform with the separate solution which has an increased concentration of the c isoform;

(d) treating the resulting combined solution so as to produce a solution which has an increased concentration of the b isoform;
and

(e) recovering the then-resulting solution which has an increased concentration of the b isoform.

51. A method of claim 50, wherein the treatment of the resulting combined solution in step (d) comprises incubation.

52. A method of claim 50, wherein the treatment of the resulting solution in step (a) comprises ion exchange chromatography.

53. A method of claim 50, which further comprises treating the solution with an increased concentration of the b isoform from step (b) or (e) so as to purify the b isoform present in the solution.

54. A method of claim 53, wherein the treatment of the solution comprises ion exchange chromatography.

55. A method of claim 54, wherein the ion exchange chromatography comprises anion exchange chromatography.

56. A method of claim 55, wherein the ion exchange chromatography further comprises cation exchange chromatography.

57. A method of increasing the yield of recovered solutions having an increased concentration of b isoform of an enzymatically-active polypeptide analog of Cu-Zn superoxide dismutase from a composition which comprises cells containing a, b and c isoforms of the polypeptide analog which comprises:

(a) treating the composition so as to separate soluble proteins present in the cells from whole cells, cellular debris and insoluble proteins so as to obtain a solu tion containing such soluble proteins, including the a, b and c isoforms;

(b) treating the resulting solution containing the soluble proteins so as to produce three separate solutions, each of which has an increased concentration of one of either of the a, b or c isoform;

(c) recovering the separate solution which has an increased concentration of the b isoform;

(d) combining the separate solution which has an increased concentration of the a isoform with the separate solution which has an increased concentration of the c isoform;

(e) treating the resulting combined solution so as to produce a solution which has an increased concentration of the b isoform;
and

(f) recovering the then-resulting solution which has an increased concentration of the b isoform.

58. A method of claim 57, wherein the treatment of the resulting combined solution in step (e) comprises incubation.

59. A method of claim 57, wherein the polypeptide analog of Cu-Zn superoxide dismutase is a polypeptide analog of human Cu-Zn superoxide dismutase.

60. A method of claim 57, wherein the treatment of the composition in step (a) comprises treating the composition so as to disrupt the cells and obtain a cellular extract therefrom, and then subjecting the resulting cellular extract to centrifugation so as to obtain the solution containing the soluble proteins.

61. A method of claim 57, wherein the treatment of the resulting solution containing the soluble proteins so as to produce three separate solutions comprises ion exchange chromatography.

62. A method of claim 57, which further comprises treating the solution with an increased concentration of the b isoform from step (c) or (f) so as to purify the b isoform present in the solution.

63. A method of claim 62, wherein the treatment of the solution comprises ion exchange chromatography.

64. A method of claim 63, wherein the ion exchange chromatography comprises anion exchange chromatography.

65. A method of claim 64, wherein the ion exchange chromatography further comprises cation exchange chromatography.