Human arginase and pegylated human arginase and the use thereof
View Patent ↗The present invention provides a site-directed mutated arginase and the preparation method thereof, and the use of said site-directed mutated arginase in preparing a medicament for treating an arginase-related disease. The present invention also provides a pegylated arginase and the preparation method thereof, and the use of said pegylated arginase in preparing a medicament for treating an arginase-related disease.
1. An arginase, wherein said arginase is human arginase I comprising an amino acid sequence of SEQ ID NO:1 comprising a mutation at position 45 of said SEQ ID NO:1; wherein said mutation is from cysteine to alanine;
wherein said arginase has activity.
2. The arginase of claim 1 , wherein said arginase further comprises a mutation at any one or two of positions 168 and 303 of SEQ ID NO:1.
3. The arginase of claim 1 , wherein said arginase has an activity of at least about 500 U/mg.
4. A pegylated arginase comprising said arginase of claim 2 , wherein said arginase is conjugated with polyethylene glycol (PEG) by pegylation modification.
5. The pegylated arginase of claim 4 , wherein the total molecular weight of said PEG molecule conjugated with each molecule of said arginase is about 20-70K.
6. The pegylated arginase of claim 4 , wherein said PEG molecule has an average molecular weight of about 2-40K.
7. The pegylated arginase of claim 4 , wherein the pegylation is achieved by covalently conjugating a PEG molecule with a moiety of said arginase using a pegylation reagent; wherein said pegylation reagent comprises pegylation reagents conjugated with ε-NH 2 of surface lysine or N-terminal α-NH 2 of the protein and pegylation reagents conjugated with thiol group or carboxyl group of amino acids of the protein; wherein said pegylation reagent is selected from a group consisting of methoxy polyethylene glycol-succinimidyl propionate (mPEG-SPA), mPEG-succinimidyl butyrate (mPEG-SBA), mPEG-succinimidyl succinate (mPEG-SS), mPEG-succinimidyl carbonate (mPEG-SC), mPEG-succinimidyl glutarate (mPEG-SG), mPEG-N-hydroxyl-succinimide (mPEG-NHS), mPEG-tresylate and mPEG-aldehyde.
8. The pegylated arginase of claim 4 , wherein any one of said cysteines at positions 168 and 303 of said SEQ ID NO:1 is mutated to alanine.
9. A pharmaceutical composition for treating an arginase-related disease comprising said pegylated arginase of claim 4 .
10. A method for treating an arginase-related disease, comprising administrating said pegylated arginase of claim 4 .
11. The arginase of claim 2 , wherein any one of said cysteines at positions 45, 168 and 303 of said SEQ ID NO:1 is mutated to alanine.
12. The arginase of claim 2 , wherein any two of said cysteines at positions 45, 168 and 303 of said SEQ ID NO:1 are mutated to alanine.
13. The arginase of claim 2 , wherein all of said cysteines at positions 45, 168 and 303 of said SEQ ID NO:1 are mutated to alanine.
14. The pegylated arginase of claim 4 , wherein any two of said cysteines at positions 45, 168 and 303 of said SEQ ID NO:1 are mutated to alanine.
15. The pegylated arginase of claim 4 , wherein all of said cysteines at positions 45, 168 and 303 of said SEQ ID NO:1 are mutated to alanine.
16. The pegylated arginase of claim 4 , wherein each of said arginase molecules is conjugated with about 4-13 PEG molecules.
17. The pegylated arginase of claim 16 , wherein each of said arginase molecules is conjugated with about 6-12 PEG molecules.
18. The pegylated arginase of claim 4 , wherein any two of said cysteines at positions 45, 168 and 303 of said SEQ ID NO:1 are mutated to alanine; said PEG molecule has an average molecular weight of about 5K; each of said arginase molecules is conjugated with about 6-12 PEG molecules.
19. The pegylated arginase of claim 4 , wherein any one of said cysteines at positions 168 and 303 of said SEQ ID NO:1 is mutated to alanine; said PEG molecule has an average molecular weight of about 5K; each of said arginase molecules is conjugated with about 6-12 PEG molecules.
20. The pegylated arginase of claim 4 , wherein the purity of said pegylated arginase is above 90%.
21. The pegylated arginase of claim 7 , wherein said pegylation reagent is methoxy polyethylene glycol-succinimidyl propionate (m PEG-SPA).
22. The pharmaceutical composition of claim 9 , wherein said disease is selected from hyperargininemia and arginine-dependent hyperplasia or tumor.
23. The method of claim 10 , wherein said disease is selected from hyperargininemia and arginine-dependent hyperplasia or tumor.