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1. (WO2007002532) CELL LINES FOR EXPRESSING ENZYME USEFUL IN THE PREPARATION OF AMIDATED PRODUCTS
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-76 - WHAT IS CLAIMED IS

1. A CHO Kl cell transfected with an expression vector for expressing peptidylglycine alpha-amidating monooxygenase.

2. The transformed cell of claim 1 wherein said expression vector has a coding region with nucleic acids encoding peptidylglycine alpha-amidating monooxygenase operably linked to a control region including a ribosome binding site, a promoter and an SV40 enhancer upstream of said promoter.

3. The transformed cell of claim 1 wherein said expression vector has a coding region with nucleic acids encoding peptidylglycine alpha-amidating monooxygenase operably linked to a control region having a ribosome binding site and, a human metallothionin HA promoter.

4. The transformed cell of claim 3 wherein said vector further includes an enhancer upstream of said promoter.

5. The transfected cell of claim 4 wherein said enhancer is an SV40 enhancer.

6. A recombinant expression vector having a coding region with nucleic acids encoding peptidylglycine alpha-amidating monooxygenase, operably linked to a control region including a ribosome binding site, a promoter and an S V40 enhancer upstream of said promoter.

7. A recombinant expression vector having a coding region with nucleic acids encoding peptidylglycine alpha-amidating monooxygenase, operably linked to -77 -a control region including a ribosome binding site and, a human metallothionin ÎȘIA promoter.

8. The vector of claim 7 wherein said vector further includes an enhancer upstream of said promoter.

9. The vector of claim 8 wherein said enhancer is an SV40 enhancer.

10. A method of preparing a cell line for the expression of peptidylglycine alpha-amidating monooxygenase, said method comprising the steps of:
(A) Transfecting potential host cells in the presence of first, second and third expression vectors, wherein said first vector includes a coding region encoding a first selectable marker, wherein said second vector includes a coding region encoding a second selectable marker, wherein said third vector includes a coding region encoding peptidylglycine alpha-amidating monooxygenase , wherein the
concentration ratio of said third vector to said first vector is at least 3:1, and wherein the concentration ratio of said third vector to said second vector is at least 3:1;
(B) Subjecting the cells resulting from step (A) to selectable pressure to select cells that have been transfected with said first vector;
(C) Subjecting the cells resulting from step (B) to selectable pressure to select cells that have been transfected with said second vector;
(D) Subjecting the cells resulting from step (C) to limiting dilution and selecting cells expressing peptidylglycine alpha-amidating monoxygenase.

11. The method of claim 10 further comprising the step of amplifying peptidylglycine alpha-amidating monooxygenase by subjecting cells resulting from step (D) to amplifying selective pressure.

12. The method of claim 10 wherein said first selectable marker is a gene encoding neomycin resistance.

-78 - 13. The method of claim 10 wherein said second selectable marker is a gene encoding dihydrofolate reductase.

14. The method of claim 11 wherein at least one of said selectable markers is a gene encoding dihydrofolate reductase and wherein said amplification is conducted in the presence of methotrexate.

15. The method of claim 10 wherein the concentration ratio of said third vector to said first vector is at least 10: 1 and the concentration ratio of said third vector to said second vector is at least 10: 1.

16. The method of claim 10 wherein said third vector includes a coding region having nucleic acids encoding peptidylglycine alpha-amidating
monooxygenase, operably linked to a control region including a ribosome binding site, a promoter and an S V40 enhancer upstream of said promoter.

17. The method of claim 10 wherein said third vector includes a coding region having nucleic acids encoding peptidylglycine alpha-amidating
monooxygenase, operably linked to a control region including a ribosome binding site, human metallothionin to a promoter and an enhancer upstream of said promoter.

18. The method of claim 17 wherein said enhancer is an SV40 enhancer.

19. The method of claim 10 wherein said potential host cells include CHO Kl cells.

20. A cell line that results from the method of claim 10.

21. A cell line comprising host cells transfected with the vector of claim 6.

-79 - 22. Peptidylglycine alpha-amidating monooxygenase expressed by the cells or cell lines of any of claims 1- 5, 20 or 21.

23. A method for the in vitro production of an amidated product, said method comprising reacting a precursor having a glycine residue, in free acid form and attached to a carbonyl group, in the presence of the peptidylglycine alpha-amidating monooxygenase of claim 22.

24. The method of claim 23 wherein said precursor is a peptide with a C-terminal glycine.

25. An amidated product resulting from the method of claim 23.

26. Cell line UGL 73-26/M.

27. Peptidylglycine alpha-amidating monooxygenase expressed by cell line UGL 73-26/M.

28. The method of claim 23 wherein said peptidylglycine alpha-amidating monooxygenase was expressed by cell line UGL 73-26/M.

29. Amidated product resulting from the method of claim 28.

30. A method of purifying peptidylglycine alpha-amidating monooxygenase following expression and secretion into culture media, wherein said method includes the steps of :
(A) Subjecting an impure sample of peptidylglycine alpha-amidating
monooxygenase to anion exchange chromatography wherein elution is isocratic;

(B) Subjecting the eluant of step (A) to hydrophobic interaction chromatography, wherein ammonium sulfate is not used and wherein elution is isocratic.

-80 -31. The method of claim 30 wherein sodium citrate is utilized to facilitate column binding in step (B).

32. A method for the in vitro production of an amidated product said method comprising:
(A) forming a hydroxylated intermediate by reacting a precursor having a glycine residue, in free acid form and attached to a carbonyl group, in the presence of peptidylglycine alpha-hydroxylating monooxygenase; and
(B) simultaneously or subsequently reacting said intermediate with either a Lewis base or peptidyl alpha-hydroxyglycine alpha-amidating lyase;
wherein said peptidylglycine alpha-hydroxylating monooxygenase has been derived by separating the peptidylglycine alpha-hydroxylating monooxygenase catalytic domain from peptidylglycine alpha-amidating monooxygenase expressed by the cells or cell lines of claims 1-5, 20 or 21.

33. The method of claim 32 wherein a Lewis base is utilized for step (B).

34. A CHO Kl cell transfected with an expression vector for expressing peptidylglycine alpha-hydroxylating monooxygenase.

35. The transformed cell of claim 34 wherein said expression vector has a coding region with nucleic acids encoding peptidylglycine alpha- hydroxylating monooxygenase operably linked to a control region including a ribosome binding site, a promoter and an S V40 enhancer upstream of said promoter.

36. The transformed cell of claim 34 wherein said expression vector has a coding region with nucleic acids encoding peptidylglycine alpha- hydroxylating monooxygenase operably linked to a control region having a ribosome binding site and, a human metallothionin IIA promoter.

-81 -37. The transformed cell of claim 36 wherein said vector further includes an enhancer upstream of said promoter.

38. The transformed cell of claim 37 wherein said enhancer is an SV40 enhancer.

39. A recombinant expression vector having a coding region with nucleic acids encoding peptidylglycine alpha- hydroxylating monooxygenase, operably linked to a control region including a ribosome binding site, a promoter and an S V40 enhancer upstream of said promoter.

40. A recombinant expression vector having a coding region with nucleic acids encoding peptidylglycine alpha-hydroxylating monooxygenase, operably linked to a control region including a ribosome binding site and, a human
metallothionin HA promoter.

41. The vector of claim 40 wherein said vector further includes an enhancer upstream of said promoter.

42. The vector of claim 41 wherein said enhancer is an SV40 enhancer.

43. A method of preparing a cell line for the expression of peptidylglycine alpha- hydroxylating monooxygenase, said method comprising the steps of:
(A) Transfecting potential host cells in the presence of first, second and third expression vectors, wherein said first vector includes a coding region encoding a first selectable marker, wherein said second vector includes a coding region encoding a second selectable marker, wherein said third vector includes a coding region encoding peptidylglycine alpha-hydroxylating monooxygenase, wherein the concentration ratio of said third vector to said first vector is at least 3:1, and wherein the concentration ratio of said third vector to said second vector is at least 3: 1;

-82 - (B) Subjecting the cells resulting from step (A) to selectable pressure to select cells that have been transfected with said first vector;
(C) Subjecting the cells resulting from step (B) to selectable pressure to select cells that have been transfected with said second vector;
(D) Subjecting the cells resulting from step (C) to limiting dilution and selecting cells expressing peptidylglycine alpha- hydroxylating monoxygenase.

44. The method of claim 43 further comprising the step of amplifying peptidylglycine alpha- hydroxylating monooxygenase by subjecting cells resulting from step (D) to amplifying selective pressure.

45. The method of claim 43 wherein said first selectable marker is a gene encoding neomycin resistance.

46. The method of claim 43 wherein said second selectable marker is a gene encoding dihydrofolate reductase.

47. The method of claim 44 wherein at least one of said selectable markers is a gene encoding dihydrofolate reductase and wherein said amplification is conducted in the presence of methotrexate.

48. The method of claims 43 wherein the concentration ratio of said third vector to said first vector is at least 10: 1 and the concentration ratio of said third vector to said second vector is at least 10: 1.

49. The method of claim 43 wherein said third vector includes a coding region having nucleic acids encoding peptidylglycine alpha-hydroxylating monoxygenase, operably linked to a control region including a ribosome binding site, a promoter and an S V40 enhancer upstream of said promoter.

-83 - 50. The method of claim 43 wherein said third vector includes a coding region having nucleic acids encoding peptidylglycine alpha-hydroxylating monooxygenase, operably linked to a control region including a ribosome binding site, human metallothionin to a promoter and an enhancer upstream of said promoter.

51. The method of claim 50 wherein said enhancer is an SV40 enhancer.

52. The method of claim 43 wherein said potential host cells include CHO Kl cells.

53. A cell line that results from any of the methods of claims 43-52.

54. A cell line comprising host cells transfected with the vector of any of claims 39-42.

55. Peptidylglycine alpha-hydroxylating monooxygenase expressed by the cells or cell lines of any of claims 34-38.

56. A method for the in vitro production of an amidated product, said method comprising reacting a precursor having a glycine residue, in free acid form and attached to a carbonyl group, in the presence of the peptidylglycine alpha-hydroxylating monooxygenase of claim 55, followed by further reaction in the presence of Lewis base or peptidyl alpha-hydroxyglycine alpha-amidating lyase.

57. The method of claim 56 wherein said precursor is a peptide with a C-terminal glycine.

58. An amidated product resulting from the method of claim 56.

-84 - 59. A method of purifying peptidylglycine alpha- hydroxylating
monooxygenase following expression and secretion into culture media, wherein said method includes the steps of :
(A) Subjecting an impure sample of peptidylglycine alpha- hydroxylating
monooxygenase to anion exchange chromatography wherein elution is isocratic;

(B) Subjecting the eluant of step (A) to hydrophobic interaction chromatography, wherein ammonium sulfate is not used and wherein elution is isocratic.

60. The method of claim 59 wherein sodium citrate is utilized to facilitate column binding in step (B).

61. A method for the in vitro production of an amidated product said method comprising:
(A) forming a hydroxylated intermediate by reacting a precursor having a glycine residue, in free acid form and attached to a carbonyl group, in the presence of peptidylglycine alpha-hydroxylating monooxygenase; and
(B) simultaneously or subsequently reacting said intermediate with either a Lewis base or peptidyl alpha-hydroxyglycine alpha-amidating lyase;
wherein said peptidylglycine alpha-hydroxylating monooxygenase has been expressed by the cells or cell lines of claims 34-38.

62. The method of claim 61 wherein step (B) utilizes a Lewis base.

63. A cell line comprising host cells transfected with the vector of claim 7.