Recombinant factor X with no glycosylation and method for preparing the same
View Patent ↗A Factor X (hereinafter referred to as “FX”) with a high activity is provided. The present invention relates to a method for efficiently preparing a recombinant, two-chain FX which comprises intervening glycosylation at such an amino acid sequence that is essential for glycosylation in FX to thereby allow for expression of a recombinant FX with no glycosylation, and the recombinant FX with no glycosylation obtained by said method.
1. A process for preparing a recombinant Factor X with inhibited glycosylation, wherein the process comprises inhibiting glycosylation of asparagine at position 181 (N181) of SEQ ID NO: 1, inhibiting glycosylation of asparagine at position 191 (N191) of SEQ ID NO: 1, or inhibiting glycosylation of asparagine at position 181 (N181) and position 191 (N191) of SEQ ID NO: 1.
2. The process according to claim 1 , wherein the process comprises inhibiting glycosylation of asparagine at position 181 (N181) of SEQ ID NO: 1.
3. The process according to claim 2 , wherein the process comprises substituting a protein comprising an amino acid other than asparagine for asparagine at position 181 (N181) of SEQ ID NO: 1.
4. The process according to claim 2 , wherein the process comprises substituting a protein comprising an amino acid other than threonine or serine for threonine at position 183 (T183) of SEQ ID NO: 1.
5. The process according to claim 1 , wherein the process comprises inhibiting glycosylation of asparagine at position 191 (N191) of SEQ ID NO: 1.
6. The process according to claim 5 , wherein the process comprises substituting a protein comprising an amino acid other than asparagine for asparagine at position 191 (N191) of SEQ ID NO: 1.
7. The process according to claim 5 , wherein the process comprises substituting a protein comprising an amino acid other than threonine or serine for threonine at position 193 (T193) of SEQ ID NO: 1.
8. The process according to claim 1 , wherein the process comprises inhibiting glycosylation of asparagine at position 181 (N181) and position 191 (N191) of SEQ ID NO: 1.
9. The process according to claim 8 , wherein the process comprises substituting a protein comprising an amino acid other than asparagine for asparagine at position 181 (N181) and position 191 (N191) of SEQ ID NO: 1.
10. The process according to claim 8 , wherein the process comprises substituting a protein comprising an amino acid other than threonine or serine for threonine at position 183 (T183) and position 193 (T193) of SEQ ID NO: 1.
11. A recombinant Factor X with inhibited glycosylation, wherein the recombinant Factor X is obtained by a process comprising inhibiting glycosylation of asparagine at position 181 (N181) of SEQ ID NO: 1, inhibiting glycosylation of asparagine at position 191 (N191) of SEQ ID NO: 1, or inhibiting glycosylation of asparagine at position 181 (N181) and position 191 (N191) of SEQ ID NO: 1.
12. The recombinant Factor X with inhibited glycosylation according to claim 11 , wherein the recombinant Factor X is obtained by a process comprising inhibiting glycosylation of asparagine at position 181 (N181) of SEQ ID NO: 1 by substituting a protein comprising an amino acid other than asparagine for asparagine at position 181 (N181) of SEQ ID NO: 1, substituting a protein comprising an amino acid other than threonine or serine for threonine at position 183 (T183) of SEQ ID NO: 1, or both.
13. A composition comprising:
the recombinant Factor X according to claim 12 ; and
a physiologically acceptable excipient.
14. A method of treating hemophilia, wherein the method comprises administering to a subject in need thereof an effective amount of the composition according to claim 13 .
15. The recombinant Factor X with inhibited glycosylation according to claim 11 , wherein the recombinant Factor X is obtained by a process comprising inhibiting glycosylation of asparagine at position 191 (N191) of SEQ ID NO: 1 by substituting a protein comprising an amino acid other than asparagine for asparagine at position 191 (N191) of SEQ ID NO: 1, substituting a protein comprising an amino acid other than threonine or serine for threonine at position 193 (T193) of SEQ ID NO: 1, or both.
16. A composition comprising:
the recombinant Factor X according to claim 15 ; and
a physiologically acceptable excipient.
17. A method of treating hemophilia, wherein the method comprises administering to a subject in need thereof an effective amount of the composition according to claim 16 .
18. The recombinant Factor X with inhibited glycosylation according to claim 11 , wherein the recombinant Factor X is obtained by a process comprising inhibiting glycosylation of asparagine at position 181 (N181) and position 191 (N191) of SEQ ID NO: 1 by: substituting a protein comprising an amino acid other than asparagine for asparagine at position 181 (N181) of SEQ ID NO: 1, substituting a protein comprising an amino acid other than threonine or serine for threonine at position 183 (T183) of SEQ ID NO: 1, or both; and substituting a protein comprising an amino acid other than asparagine for asparagine at position 191 (N191) of SEQ ID NO: 1, substituting a protein comprising an amino acid other than threonine or serine for threonine at position 193 (T193) of SEQ ID NO: 1, or both.
19. A composition comprising:
the recombinant Factor X according to claim 18 ; and
a physiologically acceptable excipient.
20. A method of treating hemophilia, wherein the method comprises administering to a subject in need thereof an effective amount of the composition according to claim 19 .
21. A process for preparing a recombinant Factor X with inhibited glycosylation, wherein the process comprises inhibiting glycosylation of threonine at position 159 (T159) of SEQ ID NO: 1, inhibiting glycosylation of threonine at position 443 (T443) of SEQ ID NO: 1, or inhibiting glycosylation of threonine at position 159 (T159) and position 443 (T443) of SEQ ID NO: 1.
22. The process according to claim 21 , wherein the process comprises inhibiting glycosylation of threonine at position 159 (T159) of SEQ ID NO: 1.
23. The process according to claim 22 , wherein the process comprises substituting an amino acid other than threonine or serine for threonine at position 159 (T159) of SEQ ID NO: 1.
24. The process according to claim 22 , wherein the process comprises substituting a protein comprising an amino acid other than threonine or serine for threonine at position 159 (T159) of SEQ ID NO: 1.
25. The process according to claim 21 , wherein the process comprises inhibiting glycosylation of threonine at position 443 (T443) of SEQ ID NO: 1.
26. The process according to claim 25 , wherein the process comprises substituting an amino acid other than threonine or serine for threonine at position 443 (T443) of SEQ ID NO: 1.
27. The process according to claim 25 , wherein the process comprises substituting a protein comprising an amino acid other than threonine or serine for threonine at position 443 (T443) of SEQ ID NO: 1.
28. The process according to claim 21 , wherein the process comprises inhibiting glycosylation of threonine at position 159 (T159) and position 443 (T443) of SEQ ID NO: 1.
29. The process according to claim 28 , wherein the process comprises substituting an amino acid other than threonine or serine for threonine at position 159 (T159) and position 443 (T443) of SEQ ID NO: 1.
30. The process according to claim 28 , wherein the process comprises substituting a protein comprising an amino acid other than threonine or serine for threonine at position 159 (T159) and position 443 (T443) of SEQ ID NO: 1.
31. A recombinant Factor X with inhibited glycosylation, wherein the recombinant Factor X is obtained by a process comprising inhibiting glycosylation of threonine at position 159 (T159) of SEQ ID NO: 1, inhibiting glycosylation of threonine at position 443 (T443) of SEQ ID NO: 1, or inhibiting glycosylation of threonine at position 159 (T159) and position 443 (T443) of SEQ ID NO: 1.
32. The recombinant Factor X with inhibited glycosylation according to claim 31 , wherein the recombinant Factor X is obtained by a process comprising inhibiting glycosylation of threonine at position 159 (T159) of SEQ ID NO: 1 by substituting an amino acid other than threonine or serine for threonine at position 159 (T159) of SEQ ID NO: 1, substituting a protein comprising an amino acid other than threonine or serine for threonine at position 159 (T159) of SEQ ID NO: 1, or both.
33. A composition comprising:
the recombinant Factor X according to claim 32 ; and
a physiologically acceptable excipient.
34. A method of treating hemophilia, wherein the method comprises administering to a subject in need thereof an effective amount of the composition according to claim 33 .
35. The recombinant Factor X with inhibited glycosylation according to claim 31 , wherein the recombinant Factor X is obtained by a process comprising inhibiting glycosylation of threonine at position 443 (T443) of SEQ ID NO: 1 by substituting an amino acid other than threonine or serine for threonine at position 443 (T443) of SEQ ID NO: 1, substituting a protein comprising an amino acid other than threonine or serine for threonine at position 443 (T443) of SEQ ID NO: 1, or both.
36. A composition comprising:
the recombinant Factor X according to claim 35 ; and
a physiologically acceptable excipient.
37. A method of treating hemophilia, wherein the method comprises administering to a subject in need thereof an effective amount of the composition according to claim 36 .
38. The recombinant Factor X with inhibited glycosylation according to claim 31 , wherein the recombinant Factor X is obtained by a process comprising inhibiting glycosylation of threonine at position 159 (T159) and position 443 (T443) of SEQ ID NO: 1 by: substituting an amino acid other than threonine or serine for threonine at position 159 (T159) of SEQ ID NO: 1, substituting a protein comprising an amino acid other than threonine or serine for threonine at position 159 (T159) of SEQ ID NO: 1, or both; and substituting an amino acid other than threonine or serine for threonine at position 443 (T443) of SEQ ID NO: 1, substituting a protein comprising an amino acid other than threonine or serine for threonine at position 443 (T443) of SEQ ID NO: 1, or both.
39. A composition comprising:
the recombinant Factor X according to claim 38 ; and
a physiologically acceptable excipient.
40. A method of treating hemophilia, wherein the method comprises administering to a subject in need thereof an effective amount of the composition according to claim 39 .