Composition, method, and kit for preparing plasmin

1. A method for preparing plasmin, the method comprising:

(a) contacting a composition comprising a plasminogen with a streptokinase immobilized on a matrix thereby converting the plasminogen to a plasmin,

wherein the streptokinase is a streptokinase mutant characterized as capable of activating plasminogen to plasmin, yet resistant to plasmin degradation relative to its corresponding wild-type streptokinase; and

wherein the streptokinase comprises an amino acid sequence having an amino acid residue other than lysine at a position corresponding to position 85, 412, or both positions, as shown in SEQ ID NO:1; and

(b) purifying the plasmin by a method comprising contacting the composition with a plasmin-binding matrix so that the plasmin is retained by the plasmin-binding matrix, the plasmin-binding matrix having a molecule disposed thereon having affinity for the plasmin.

2. The method of claim 1 , wherein the streptokinase comprises the amino acid sequence shown as amino acid residues 27 through 440 of SEQ ID NO:2.

3. The method of claim 1 , wherein the streptokinase, optionally, further comprises polar or charged amino acid residues comprising lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, tryosine or cysteine at one or more positions corresponding to positions 406-410 in SEQ ID NO:1.

4. The method of claim 1 , wherein the amino acid residue is glutamine or asparagine.

5. The method of claim 2 , wherein the streptokinase, optionally, further comprises polar or charged amino acid residues comprising lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, tryosine or cysteine at one or more positions corresponding to positions 406-410 in SEQ ID NO:1.

6. The method of claim 1 , wherein the streptokinase is recombinant.

7. The method of claim 1 , wherein the streptokinase comprises an affinity tag.

8. The method of claim 7 , wherein the affinity tag comprises polyhistidine, thioredoxin, or glutathione-S-transferase (GST).

9. The method of claim 7 , wherein the affinity tag comprises polyhistidine.

10. The method of claim 1 , wherein the streptokinase is immobilized on a matrix comprising immobilized metal ion adsorption chromatography.

11. The method of claim 1 , wherein the plasma-binding matrix is benzamidine-coupled to crosslinked beaded agarose.

12. The method of claim 1 , wherein the plasminogen is plasma-derived.

13. The method of claim 1 , wherein the plasminogen is recombinant.

14. A method for preparing plasmin, the method comprising:

(a) contacting a composition comprising a plasminogen with a streptokinase immobilized on a matrix thereby converting the plasminogen to a plasmin,

wherein the streptokinase is a streptokinase mutant characterized as capable of activating plasminogen to plasmin, yet resistant to plasmin degradation relative to its corresponding wild-type streptokinase;

wherein the streptokinase comprises an amino acid sequence having an amino acid residue other than lysine at a position corresponding to position 85, 412, or both positions, as shown in SEQ ID NO:1; and

wherein the streptokinase comprises a polyhistidine affinity tag; and

(b) purifying the plasmin by a method comprising contacting the composition with a plasmin-binding matrix so that the plasmin is retained by the plasmin-binding matrix;

wherein the plasma-binding matrix is benzamidine-coupled to crosslinked beaded agarose.

15. The method of claim 14 , wherein the streptokinase comprises the amino acid sequence shown as amino acid residues 27 through 440 of SEQ ID NO:2.

16. The method of claim 14 , wherein the streptokinase, optionally, further comprises polar or charged amino acid residues comprising lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, tryosine or cysteine at one or more positions corresponding to positions 406-410 in SEQ ID NO:1.

17. The method of claim 14 , wherein the amino acid residue is glutamine or asparagine.

18. The method of claim 15 , wherein the streptokinase, optionally, further comprises polar or charged amino acid residues comprising lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, tryosine or cysteine at one or more positions corresponding to positions 406-410 in SEQ ID NO:1.

19. The method of claim 14 , wherein the plasminogen is plasma-derived.

20. The method of claim 14 , wherein the plasminogen is recombinant.