Variant, recombinant beta-glucocerebrosidase proteins with increased stability and increased retained catalytic activity
Described herein are variant, recombinant β-glucocerebrosidase proteins characterized as having increased stability relative to recombinant wild-type β-glucocerebrosidase. Also provided herein are variant, recombinant β-glucocerebrosidase proteins characterized as retaining more catalytic activity relative to recombinant wild-type β-glucocerebrosidase. Further described herein are variant, recombinant β-glucocerebrosidase proteins that can have amino acid variations at one or more of the following positions: 316, 317, 321 and 145. Methods of making the variant, recombinant β-glucocerebrosidase proteins are also described as well as methods of treating patients having lysosomal storage diseases.
1. A variant, recombinant human β-glucocerebrosidase protein, characterized as having increased stability relative to human wild-type, recombinant β-glucocerebrosidase, wherein the protein is derived from SEQ ID NO: 1 by a substitution selected from the group consisting of F316A, L317F, K321V, K321A, H145L and H145F.
2. The variant, recombinant β-glucocerebrosidase protein of claim 1 wherein the increased stability is monitored at conditions of about neutral pH and about 37° C. over a period of about three hours.
3. The variant, recombinant β-glucocerebrosidase protein of claim 1 wherein the increased stability is inside cells.
4. The variant, recombinant β-glucocerebrosidase protein of claim 1 wherein the increased stability is characterized by reduced proteolytic degradation.
5. The variant, recombinant β-glucocerebrosidase protein of claim 1 wherein the increased stability is in buffer solutions during protein purification.
6. A variant, recombinant human β-glucocerebrosidase protein, characterized as retaining more catalytic activity relative to human wild-type, recombinant β-glucocerebrosidase, wherein the protein is derived from SEQ ID NO: 1 by a substitution selected from the group consisting of F316A, L317F, K321V, K321A, H145L and H145F.
7. The variant, recombinant β-glucocerebrosidase protein of claim 6 wherein more catalytic activity is retained at conditions of about neutral pH and about 37° C.
8. The variant, recombinant β-glucocerebrosidase protein of claim 6 wherein more catalytic activity is retained inside cells.
9. The variant, recombinant β-glucocerebrosidase protein according to claim 1 , wherein the protein comprises amino acid variations selected from the group consisting of F316A and L317F; F316A, L317F, and K321N; F316A, L317F, and K321A; F316A, L317F, and K321V; H145L and K321N; H145L and K321A; H145L and K321V; H145L, F316A, and L317F.
10. A method of making the variant, recombinant human β-glucocerebrosidase protein according to claim 1 , the method comprising:
(a) expressing the variant, recombinant β-glucocerebrosidase protein; and
(b) purifying the variant, recombinant β-glucocerebrosidase protein.
11. A composition comprising the variant, recombinant β-glucocerebrosidase protein of any one of claim 1 or 9 and a pharmaceutically acceptable carrier.
12. A composition comprising the variant, recombinant β-glucocerebrosidase protein of any one of claim 1 or 9 and a pharmaceutically acceptable buffer.
13. A method for treating a lysosomal storage disease, comprising administering to a human subject in need thereof the variant, recombinant human β-glucocerebrosidase protein of claim 1 .
14. The method for treating a lysosomal storage disease of claim 13 , wherein the variant, recombinant β-glucocerebrosidase protein comprises one of the following amino acid variations F316A and L317F; K321A; K321V; H145L; H145F; F316A, L317F, and K321N; F316A, L317F, and K321A; F316A, L317F, and K321V; H145L, F316A, and L317F; H145L and K321N; H145L and K321A; H145L and K321V; H145L, F316A, L317F and K321N; H145L, F316A, L317F and K321A; H145L, F316A, L317F and K321V.
15. The method of claim 14 wherein the lysosomal storage disease is Gaucher disease.
16. The method of claim 15 , wherein the administration is intravenous infusion.
17. The method of claim 15 , wherein the administration is intraperitoneal injection.
18. A compound comprising a variant, recombinant β-glucocerebrosidase protein characterized as having any one of the following amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.
19. A composition comprising:
a pharmaceutically acceptable carrier; and
a variant, recombinant β-glucocerebrosidase protein characterized as having any one of the following amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.
20. A method of making a composition, the method comprising adding to a pharmaceutically acceptable carrier a flail variant, recombinant β-glucocerebrosidase protein characterized as having any one of the following amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.
21. A method of making a nucleic acid encoding a variant, recombinant β-glucocerebrosidase protein, the method comprising:
generating a DNA plasmid with an oligonucleotide primer or synthetic DNA minigene encoding a fragment of β-glucocerebrosidase comprising any one of the following amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.
22. A variant, recombinant β-glucocerebrosidase protein derived from SEQ ID NO: 1 by a substitution at one or more of the following positions: 316, 317, 321 and 145, wherein the substitution at 321 is selected from K321V and K321A, and wherein the variant, recombinant β-glucocerebrosidase protein has increased stability relative to human wild-type, recombinant β-glucocerebrosidase.
23. A composition comprising the variant, recombinant β-glucocerebrosidase protein of claim 22 and a pharmaceutically acceptable carrier.
24. A method for treating a lysosomal storage disease, the method comprising:
administering to a human subject in need thereof the variant, recombinant human β-glucocerebrosidase protein of claim 22 .