Compositions and methods for the preparation of human growth hormone glycosylation mutants
View Patent ↗The present invention relates to mutants of human growth hormone, which contain newly introduced N-linked or O-linked glycosylation site(s), such that these recombinantly produced polypeptides have glycosylation patterns distinctly different from that of the naturally occurring human growth hormone. The polynucleotide coding sequences for the mutants, expression cassettes comprising the coding sequences, cells expressing the mutants, and methods for producing the mutants are also disclosed. Further disclosed are pharmaceutical compositions comprising the mutants and method for using the mutants.
1. An isolated human growth hormone polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 except for the presence of at least one O-linked glycosylation site not present in SEQ ID NO: 1 or SEQ ID NO: 2, wherein the O-linked glycosylation site is a serine or threonine residue, and wherein the O-linked glycosylation site is present at a site selected from the group consisting of (a) proximate a proline residue located at position 2, 5, 37, 48, 59, 89, or 133 of SEQ ID NO: 1 or SEQ ID NO: 2, (b) the amino acid sequence surrounding position 140 of SEQ ID NO: 1 or SEQ ID NO: 2, and (c) the amino acid sequence at the C-terminus of SEQ ID NO: 1 or SEQ ID NO: 2.
2. The isolated human growth hormone polypeptide of claim 1 , wherein the O-linked glycosylation site is present at a site proximate a proline residue located at position 2, 5, 37, 48, 59, 89, or 133 of SEQ ID NO: 1 or SEQ ID NO: 2.
3. The isolated human growth hormone polypeptide of claim 1 , comprising the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, 7, 8, or 9.
4. The isolated human growth hormone polypeptide of claim 1 , comprising a plurality of O-linked glycosylation sites not present SEQ ID NO: 1 or SEQ ID NO: 2.
5. The isolated human growth hormone polypeptide of claim 1 , comprising a water-soluble polymer attached to the glycosylation site through a glycosyl linker.
6. The isolated human growth hormone polypeptide of claim 5 , wherein said glycosyl linker is an intact glycosyl linker.
7. A composition comprising the isolated human growth hormone polypeptide of claim 1 and a carrier therefor.
8. A method of making a glycosylated human growth hormone polypeptide comprising:
a) recombinantly producing the human growth hormone polypeptide of claim 1 ; and
b) glycosylating said human growth hormone polypeptide at the O-linked glycosylation site.
9. The method of claim 8 , wherein step b) comprises enzymatically glycosylating said human growth hormone polypeptide with a modified sugar at the O-linked glycosylation site.
10. The method of claim 9 , wherein the sugar is modified with a poly(ethylene glycol) or m-poly(ethylene glycol).
11. The method of claim 9 , wherein the O-linked glycosylation site is present at a site proximate a proline residue located at position 2, 5, 37, 48, 59, 89, or 133 of SEQ ID NO: 1 or SEQ ID NO: 2.
12. The method of claim 9 , wherein said human growth hormone polypeptide comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, 7, 8, or 9.
13. The method of claim 9 , wherein said human growth hormone polypeptide comprises a plurality of O-linked glycosylation sites not present in SEQ ID NO: 1 or SEQ ID NO: 2.
14. The method of claim 9 , wherein said human growth hormone polypeptide comprises a water-soluble polymer attached to the glycosylation site through a glycosyl linker.
15. The method of claim 14 , wherein said glycosyl linker is an intact glycosyl linker.
16. The method of claim 8 , wherein the O-linked glycosylation site is present at a site proximate a proline residue located at position 2, 5, 37, 48, 59, 89, or 133 of SEQ ID NO: 1 or SEQ ID NO: 2.
17. The method of claim 8 , wherein said human growth hormone polypeptide comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, 7, 8, or 9.
18. The method of claim 8 , wherein said human growth hormone polypeptide comprises a plurality of O-linked glycosylation sites not present SEQ ID NO: 1 or SEQ ID NO: 2.
19. The method of claim 8 , wherein said human growth hormone polypeptide comprises a water-soluble polymer attached to the glycosylation site through a glycosyl linker.
20. The method of claim 19 , wherein said glycosyl linker is an intact glycosyl linker.
21. The isolated human growth hormone polypeptide of claim 1 , wherein the isolated human growth hormone polypeptide is glycosylated.
22. The isolated human growth hormone polypeptide of claim 2 , wherein the isolated human growth hormone polypeptide is glycosylated.
23. The isolated human growth hormone polypeptide of claim 3 , wherein the isolated human growth hormone polypeptide is glycosylated.
24. The isolated human growth hormone polypeptide of claim 4 , wherein the isolated human growth hormone polypeptide is glycosylated.
25. A composition comprising the isolated human growth hormone polypeptide of claim 2 and a carrier therefor.
26. A composition comprising the isolated human growth hormone polypeptide of claim 3 and a carrier therefor.
27. A composition comprising the isolated human growth hormone polypeptide of claim 4 and a carrier therefor.
28. A composition comprising the isolated human growth hormone polypeptide of claim 5 and a carrier therefor.
29. A composition comprising the isolated human growth hormone polypeptide of claim 6 and a carrier therefor.
30. A composition comprising the isolated human growth hormone polypeptide of claim 21 and a carrier therefor.
31. A composition comprising the isolated human growth hormone polypeptide of claim 22 and a carrier therefor.
32. A composition comprising the isolated human growth hormone polypeptide of claim 23 and a carrier therefor.
33. A composition comprising the isolated human growth hormone polypeptide of claim 24 and a carrier therefor.