Modified trypsin and production technology

Claims

1. A modified trypsin containing modified lysine residues, characterized in that it is modified by the simultaneous introduction of two different modifications, one of which is a high-molecular agent and the other a low-molecular group.

2. The product according to claim 1, characterized in that when two modifications are introduced into trypsin simultaneously, ε-PEG-lysine (high-molecular modification) and ε-dimethyl-lysine (low-molecular modification) are formed. The range of PEG residues after modification is: 6-12. The range of modified ε-dimethyl groups of lysine depends on the degree of modification of trypsin with PEG residues and can vary: 2-8.

3. The product according to claim 1, characterized in that when two modifications are introduced into trypsin simultaneously, ε-PEG-lysine (high-molecular modification) and ε-acetyl-lysine (low-molecular modification) are formed. The range of PEG residues after modification is: 6-12. The range of modified ε-acyl groups of lysine depends on the degree of modification of trypsin with PEG residues and can vary: 2-8.

4. The product according to claim 1, characterized in that when two modifications are introduced into trypsin simultaneously, ε-dextran-lysine (high-molecular modification) and ε-dimethyl-lysine (low-molecular modification) are formed. The range of dextran-2000 residues after modification is: 6-12. The range of modified ε-acyl groups of lysine depends on the degree of modification of trypsin with dextran residues and can vary: 2-8.

5. The product according to claim 1, characterized in that when two modifications are introduced into trypsin simultaneously, ε-dextran-lysine (high-molecular modification) and ɛ-acetyl-lysine (low-molecular modification) are formed. The range of dextran-2000 residues after modification is: 6-12. The range of modified ε-acyl groups of lysine depends on the degree of modification of trypsin with dextran residues and can vary: 2-8.

6. The product according to claim 1, characterized in that when two modifications are introduced into trypsin simultaneously, ε-(maleic acid copolymer)-lysine (high-molecular modification) and ɛ-dimethyl-lysine (low-molecular modification) are formed. The range of maleic acid copolymer residues after modification is: 6-12. The range of modified ε-dimethyl groups of lysine depends on the degree of modification of trypsin with dextran residues and can vary: 2-8.

7. The product according to claim 1, characterized in that when two modifications are introduced into trypsin simultaneously, ε-(maleic acid copolymer)-lysine (high-molecular modification) and ɛ-acetyl-lysine (low-molecular modification) are formed. The range of maleic acid copolymer residues after modification is: 6-12. The range of modified ε-dimethyl groups of lysine depends on the degree of modification of trypsin with dextran residues and can vary: 2-8.

8. The product according to claim 1, characterized in that when two modifications are introduced into trypsin simultaneously, ε-PEG-lysine (high-molecular modification) and ε-citraconyl-lysine (low-molecular modification) are formed. The range of PEG residues after modification is: 6-12. The range of modified ε-citraconic acyl groups of lysine depends on the degree of modification of trypsin with PEG residues and can vary: 2-8.

9. The product according to claim 1, characterized in that when two modifications are introduced into trypsin simultaneously, ε-(maleic acid copolymer)-lysine (high-molecular modification) and ε-citraconyl-lysine (low-molecular modification) are formed. The range of maleic acid copolymer residues after modification is: 6-12. The range of modified ɛ-citraconic acyl groups of lysine depends on the degree of modification of trypsin with PEG residues and can vary: 2-8.

10. The product according to claim 1, characterized in that when two modifications are introduced into trypsin simultaneously, ε-dextran-lysine (high-molecular modification) and ε-citraconyl-lysine (low-molecular modification) are formed. The range of dextran-2000 residues after modification is: 6-12. The range of modified ε-citraconic acyl groups of lysine depends on the degree of modification of trypsin with PEG residues and can vary: 2-8.

11. A technology for modifying trypsin molecules, characterized in that the side amino groups of lysine are sequentially modified, first with a high-molecular-weight modifying agent, then with a low-molecular-weight chemical group, after which the modified trypsin is purified using affinity chromatography.

12. The method according to paragraphs 2 and 11, characterized in that the modification is carried out in a borate buffer with a pH of 6.5-10.0 (optionally with the addition of benzamidine hydrochloride or soybean trypsin inhibitor at a concentration of 1-3 mg / ml) at a temperature of 4-25°C, high-molecular modification is carried out using a modifying agent based on PEG at a concentration of 6-7 mg / ml and involves incubating the reaction mixture for 20-30 minutes; Low-molecular modification is carried out by adding formaldehyde to a final concentration of 30-50 mM and a reducing agent (sodium borohydride, sodium cyanoborohydride or 2-picoline borane) to a final concentration of 15-20 mM, incubating the reaction mixture for 10-40 minutes and stopping the reaction by acidifying the medium or adding sodium bicarbonate to a final concentration of 200-250 mM.

13. The method according to paragraphs 3 and 11, characterized in that the modification is carried out in a borate buffer with a pH of 6.5-10.0 at a temperature of 4-25°C, high-molecular modification is carried out using a modifying agent based on PEG at a concentration of 6-7 mg / ml and involves incubating the reaction mixture for 20-30 minutes; low-molecular modification is carried out by adding N-hydroxysuccinimide acetate at a concentration of 0.2-0.25 mg / ml in DMSO and incubating the reaction mixture for 10-60 minutes.

14. The method according to paragraphs 8 and 11, characterized in that the modification is carried out in a borate buffer with a pH of 6.5-10.0 at a temperature of 4-25°C, high-molecular modification is carried out using a modifying agent based on PEG at a concentration of 6-7 mg / ml and involves incubating the reaction mixture for 20-30 minutes; low-molecular modification is carried out by adding 5 aliquots of 10 mg of citraconic anhydride at intervals of 15 minutes and incubating the reaction mixture for 1-2 hours.

15. The method according to paragraphs 4, 11, characterized in that the modification is carried out in a potassium phosphate buffer with a pH of 6.0-10.0 at a temperature of 4-25°C; high-molecular modification is carried out using aminated dextran at a concentration of 20-60 mg / ml and involves incubating the reaction mixture for 1 hour, adding 30 mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and repeated incubation for 1 hour; Low-molecular modification is carried out by adding formaldehyde to a final concentration of 10-30 mM and a reducing agent (sodium cyanoborohydride, sodium borohydride or 2-picoline borane) to a final concentration of 10-20 mM, incubating the reaction mixture for 10-40 minutes and stopping the reaction by acidifying the medium or adding sodium bicarbonate to a final concentration of 200-250 mM.

16. The method according to paragraphs 5 and 11, characterized in that the modification is carried out in a potassium phosphate buffer with a pH of 6.0-10.0 at a temperature of 4-25°C; high-molecular modification is carried out using aminated dextran at a concentration of 20-60 mg / ml and involves incubating the reaction mixture for 1 hour, adding 30 mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and repeated incubation for 1 hour; low-molecular modification is carried out by adding N-hydroxysuccinimide acetate at a concentration of 0.15-0.2 mg / ml in DMSO and incubating the reaction mixture for 10-60 minutes.

17. The method according to paragraphs 10 and 11, characterized in that the modification is carried out in a potassium phosphate buffer with a pH of 6.0-10.0 at a temperature of 4-25°C; high-molecular modification is carried out using aminated dextran at a concentration of 20-60 mg / ml and involves incubating the reaction mixture for 1 hour, adding 30 mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and repeated incubation for 1 hour; low-molecular modification is carried out by adding 5 aliquots of 10 mg of citraconic anhydride at intervals of 15 minutes and incubating the reaction mixture for 1-2 hours.

18. The method according to paragraphs. 6, 11, characterized in that the modification is carried out in a phosphate buffer with a pH of 6.5-10.0 at 4-25 °C; the high-molecular modifying agent is a product obtained by adding 300 μl of 120 mM N-hydroxysuccinimide and 300 μl of 300 mM N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride to a solution of poly(methyl vinyl ether-alt-maleic anhydride) with a concentration of 4 mg / ml and incubating the mixture for 1 hour at room temperature, with stirring; to carry out the high-molecular modification, 1 volume of a trypsin solution with a concentration of 1 mg / ml is added to 8 volumes of the resulting solution, the mixture is incubated at 4-25 °C for 2 hours;Low molecular weight modification is carried out by adding formaldehyde to a final concentration of 10-30 mM and a reducing agent (sodium cyanoborohydride, sodium borohydride or 2-picoline borane) to a final concentration of 10-20 mM, incubating the reaction mixture for 10-40 minutes and stopping the reaction by acidifying the medium or adding sodium bicarbonate to a final concentration of 200-250 mM.

19. The method according to paragraphs. 7, 11, characterized in that the modification is carried out in a phosphate buffer with a pH of 6.5-10.0 at 4-25 ° C; the high-molecular modifying agent is a product obtained by adding 300 μl of 120 mM N-hydroxysuccinimide and 300 μl of 300 mM N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride to a solution of poly(methyl vinyl ether-alt-maleic anhydride) with a concentration of 4 mg / ml and incubating the mixture for 1 hour at room temperature, with stirring; to carry out high-molecular modification, 1 volume of trypsin solution with a concentration of 1 mg / ml is added to 8 volumes of the resulting solution, the mixture is incubated at 4-25 ° C for 2 hours; Low molecular weight modification is achieved by adding N-hydroxysuccinimide acetate at a concentration of 0.15-0.2 mg / ml in DMSO and incubating the reaction mixture for 10-60 minutes.

20. The method according to paragraphs. 9, 11, characterized in that the modification is carried out in a phosphate buffer with a pH of 6.5-10.0 at 4-25 ° C; the high-molecular modifying agent is a product obtained by adding 300 μl of 120 mM N-hydroxysuccinimide and 300 μl of 300 mM N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride to a solution of poly(methyl vinyl ether-alt-maleic anhydride) with a concentration of 4 mg / ml and incubating the mixture for 1 hour at room temperature, with stirring; to carry out high-molecular modification, 1 volume of trypsin solution with a concentration of 1 mg / ml is added to 8 volumes of the resulting solution, the mixture is incubated at 4-25 ° C for 2 hours; Low molecular weight modification is carried out by adding 5 aliquots of 10 mg of citraconic anhydride at 15 minute intervals and incubating the reaction mixture for 1-2 hours.