Method of making cysteine depleted IL-6 muteins
| Title: | Method of making cysteine depleted IL-6 muteins |
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| Patent Number: | 5,545,537 |
| Publication Date: | August 13, 1996 |
| Appl. No: | 08/209,182 |
| Application Filed: | March 10, 1994 |
| Abstract: | Muteins of IL-6 and truncated IL-6 are prepared by recombinant DNA techniques. In the muteins, the cysteine residues that occur at positions, or at positions corresponding to positions, 45 and 51 of mature, native IL-6 have been replaced by other amino acids. The cysteine residues that occur at positions, or at positions corresponding to positions, 74 and 84 are retained. The molecule has biological activity that is at least comparable to that of native IL-6. |
| Inventors: | Skelly, Susan M. (Dunellen, NJ); Tackney, Charles T. (Brooklyn, NY); Snouwaert, John N. (Carrboro, NC); Fowlkes, Dana M. (Chapel Hill, NC) |
| Assignees: | The Trustees of Princeton University (Princeton, NJ) |
| Claim: | What we claim is |
| Claim: | 1. A method of converting a nucleic acid molecule encoding native, full length IL-6 into a nucleic acid molecule encoding a mutein with activity at least comparable to that of native IL-6 comprising the step of replacing the codon for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 with codons for other amino acid residues and retaining the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84. |
| Claim: | 2. The method of claim 1 wherein the other amino acids at positions, or at positions corresponding to positions, 45 and 51 are neutral amino acids. |
| Claim: | 3. The method of claim 1 wherein the other amino acids at positions, or at positions corresponding to positions, 45 and 51 are both serine residues. |
| Claim: | 4. A method of converting a nucleic acid molecule encoding an IL-6 molecule into a nucleic acid molecule encoding a mutein with activity at least comparable to that of native IL-6 comprising the step of replacing the codon for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 with codons for the serine residues and retaining the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84. |
| Claim: | 5. A method of converting a nucleic acid molecule encoding IL-6 into a nucleic acid molecule encoding a mutein with activity at least comparable to that of native IL-6 comprising the steps of replacing the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 with other amino acid residues; retaining the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84; and removing the codons for from 1 to 28 terminal amino acids. |
| Claim: | 6. The method of claim 5 wherein the other amino acids at positions, or at positions corresponding to positions, 45 and 51 are neutral amino acids. |
| Claim: | 7. The method of claim 5 wherein the other amino acids at positions, or at positions corresponding to positions, 45 and 51 are both serine residues. |
| Claim: | 8. The method of claim 5 wherein the 22 N-terminal amino acids are removed. |
| Claim: | 9. The method of claim 5 wherein the N-terminal alanine residue is lacking. |
| Claim: | 10. A method of converting a nucleic acid molecule encoding IL-6 into a nucleic acid molecule encoding a mutein with activity at least comparable to that of native IL-6 comprising the steps of replacing the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 with serine residues; retaining the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84; and removing the codons for the N-terminal 22 amino acids. |
| Claim: | 11. A method of converting a nucleic acid molecule encoding native IL-6 into a nucleic acid molecule encoding a mutein with activity at least comparable to that of native IL-6 comprising the steps of replacing the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 with serine residues; retaining the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84; and removing the codons for the N-terminal alanine residue. |
| Claim: | 12. A method of preparing a protein having biological activity at least comparable to that of native IL-6 comprising the steps of |
| Claim: | A. Preparing a nucleic acid molecule encoding a mutein of IL-6 wherein the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by codons for other amino acids, and the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained, and |
| Claim: | B. Expressing the mutein in a suitable host cell and |
| Claim: | C. Recovering said expressed mutein. |
| Claim: | 13. The method of claim 12 wherein the host cell is an E. coli cell. |
| Claim: | 14. The method of claim 12 wherein the other amino acids at positions, or at positions corresponding to positions, 45 and 51 are neutral amino acids. |
| Claim: | 15. The method of claim 12 wherein the other amino acids at positions, or at positions corresponding to positions, 45 and 51 are both serine residues. |
| Claim: | 16. A method of preparing a protein having biological activity at least comparable to that of native IL-6 comprising the steps of |
| Claim: | A. Preparing a nucleic acid molecule encoding a mutein of IL-6 wherein the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by codons for serine residues and the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained, and |
| Claim: | 17. The method of claim 16 wherein the host cell is an E. coli cell. |
| Claim: | 18. A method of preparing a protein having biological activity at least comparable to that of native IL-6 comprising the steps of |
| Claim: | A. Preparing a nucleic acid molecule encoding a mutein of IL-6 wherein the codon for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by codons for other amino acids; the codons for cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained; and the codons for 1 to 28 N-terminal amino acids are lacking, and |
| Claim: | 19. The method of claim 18 wherein the host cell is an E. coli cell. |
| Claim: | 20. The method of claim 18 wherein the codons for the other amino acids at positions, or at positions corresponding to positions, 45 and 51 are codons for neutral amino acids. |
| Claim: | 21. The method of claim 18 wherein the codons for the other amino acids at positions, or at positions corresponding to positions, 45 and 51 are codons for serine residues. |
| Claim: | 22. The method of claim 18 wherein the codon for the 22 N-terminal amino acids are lacking. |
| Claim: | 23. The method of claim 18 wherein the codon for the N-terminal alanine is lacking. |
| Claim: | 24. A method of preparing a protein having biological activity at least comparable to that of native IL-6 comprising the steps of |
| Claim: | A. Preparing a nucleic acid molecule encoding a mutein of IL-6 wherein the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by codons for serine residues; the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained; and the codons for the 22 N-terminal amino acids are lacking, and |
| Claim: | 25. A method preparing a protein having biological activity at least comparable to that of native IL-6 comprising the steps of |
| Claim: | A. Preparing a nucleic acid molecule encoding a mutein of IL-6 wherein the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by codons for serine residues; the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained; and the codon for the N-terminal alanine residue is lacking, and |
| Claim: | 26. The method of claim 24 or claim 25 wherein the host cell is an E. coli cell. |
| Claim: | 27. A nucleic acid molecule encoding a mutein of IL-6 wherein the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by codons for other amino acids, and the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained. |
| Claim: | 28. The nucleic acid molecule of claim 27 wherein the other amino acids at positions, or at positions corresponding to positions, 45 and 51 are neutral amino acids. |
| Claim: | 29. The nucleic acid molecule of claim 27 wherein the other amino acids at positions, or at positions corresponding to positions, 45 and 51 are both serine residues. |
| Claim: | 30. A nucleic acid molecule encoding a mutein of IL-6 wherein the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by codons for serine residues, and the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained. |
| Claim: | 31. A nucleic acid molecule encoding a mutein of IL-6 wherein the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by codons for other amino acids; the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained; and the codons for 1 to 28 terminal amino acids are lacking. |
| Claim: | 32. The nucleic acid molecule of claim 31 wherein the other amino acids at positions, or at positions corresponding to positions, 45 and 51 of the mutein are neutral amino acids. |
| Claim: | 33. The nucleic acid molecule of claim 31 wherein the other amino acids at positions, or at positions corresponding to positions, 45 and 51 of the mutein are both serine residues. |
| Claim: | 34. The nucleic acid molecule of claim 31 wherein the 22 N-terminal amino acids of the mutein are lacking. |
| Claim: | 35. The nucleic acid molecule of claim 31 wherein the N-terminal alanine residue of the mutein is lacking. |
| Claim: | 36. A nucleic acid molecule encoding a mutein of IL-6 wherein the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by a codon for serine residues; the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained; and the codons for the 22 N-terminal amino acids are lacking. |
| Claim: | 37. A nucleic acid molecule encoding a mutein of IL-6 wherein the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by codons for serine residues; the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained; and the codons for the 22 N-terminal amino acids are lacking. |
| Claim: | 38. A nucleic acid molecule encoding a mutein of IL-6 wherein the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by codons for serine residues; the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained; and the codon for the N-terminal alanine residue is lacking. |
| Claim: | 39. A nucleic acid molecule encoding a mutein of IL-6 wherein the codon for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by codons for serine residues; the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained; and the codon for the N-terminal alanine residue is lacking. |
| Claim: | 40. A host cell comprising a nucleic acid molecule encoding a mutein of IL-6 wherein the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by codons for other amino acids, and the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained. |
| Claim: | 41. The host cell of claim 40 wherein the other amino acids at positions, or at positions corresponding to positions, 45 and 51 are neutral amino acids. |
| Claim: | 42. The host cell of claim 40 wherein the other amino acids at positions, or at positions corresponding to positions, 45 and 51 are both serine residues. |
| Claim: | 43. A host cell comprising a nucleic acid molecule encoding a mutein of IL-6 wherein the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by codons for serine residues, and the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained. |
| Claim: | 44. A host cell comprising a nucleic acid molecule encoding a mutein of IL-6 wherein the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by codons for other amino acids; the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained; and the codons for 1 to 28 terminal amino acids are lacking. |
| Claim: | 45. The host cell of claim 44 wherein the other amino acids at positions, or at positions corresponding to positions, 45 and 51 of the mutein are neutral amino acids. |
| Claim: | 46. The host cell of claim 44 wherein the other amino acids at positions, or at positions corresponding to positions, 45 and 51 of the mutein are both serine residues. |
| Claim: | 47. The host cell of claim 44 wherein the 22 N-terminal amino acids of the mutein are lacking. |
| Claim: | 48. The host cell of claim 44 wherein the N-terminal alanine residue of the mutein is lacking. |
| Claim: | 49. A host cell comprising a nucleic acid molecule encoding a mutein of IL-6 wherein the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by a codon for serine residues; the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained; and the codons for the 22 N-terminal amino acids are lacking. |
| Claim: | 50. A host cell comprising a nucleic acid molecule encoding a mutein of IL-6 wherein the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by codons for serine residues; the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained; and the codons for the 22 N-terminal amino acids are lacking. |
| Claim: | 51. A host cell comprising a nucleic acid molecule encoding a mutein of IL-6 wherein the codons for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by codons for serine residues; the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained; and the codon for the N-terminal alanine residue is lacking. |
| Claim: | 52. A host cell comprising a nucleic acid molecule encoding a mutein of IL-6 wherein the codon for the cysteine residues at positions, or at positions corresponding to positions, 45 and 51 of native human IL-6 have each been replaced by codons for serine residues; the codons for the cysteine residues at positions, or at positions corresponding to positions, 74 and 84 have been retained; and the codon for the N-terminal alanine residue is lacking. |
| Current U.S. Class: | 435/6952; 4351/723; 43525/233; 4353/201; 935/22; 935/52; 935/72; 536/235 |
| Current International Class: | C12N 1524; C07K 1454 |
| Patent References Cited: | 4853332 August 1989 Mark et al. |
| Other References: | Snouwaert et al., Journal of Immunology 146, 585-591 (1991). Brakenhoff et al., Journal of Immunology 145, 561-568 (1990). Clogston et al., Archives of Biochemistry and Biophysics 272, 144-151 (1989). Brakenhoff et al., Journal of Immunology 143, 1175-1182 (1989). Brakenhoff et al., Journal of Immunology 139, 4116-4121 (1987). Asagoe et al., Biotechnology 6, 806-809 (1988). Yasueda et al., Biotechnology 8, 1036-1040 (1990). "Altered Sites in vitro Mutagenesis System Technical Manual," Promega Corporation. Sugasarawa et al., Biotechnology 6, 895-902 (1988). |
| Primary Examiner: | Draper, Garnette D. |
| Assistant Examiner: | Mertz, Prema |
| Attorney, Agent or Firm: | Mattlin, Laura W. Feit, Irving N. Gallagher, Thomas C. |
| Accession Number: | edspgr.05545537 |
| Database: | USPTO Patent Grants |
| Language: | English |
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