Nucleic acid construct for expression of alpha-galactosidase in plants and plant cells
Nucleic acid expression constructs are provided and, more particularly, nucleic acid constructs for expression of human alpha-galactosidase in plant cells, cells expressing the nucleic acid construct, producing the human alpha-galactosidase and uses thereof.
1. A nucleic acid expression construct, the construct comprising a nucleic acid sequence encoding a human α-galactosidase protein, wherein said human α-galactosidase protein is translationally fused at the N-terminal to an Arabidopsis ABPI endoplasmic reticulum targeting signal peptide and wherein said human α-galactosidase protein is translationally fused at the C-terminal to an endoplasmic reticulum retention signal peptide, and wherein said nucleic acid sequence is codon-optimized for plant expression.
2. The nucleic acid construct of claim 1 , wherein said nucleic acid sequence comprises SEQ ID NO: 22.
3. The nucleic acid construct of claim 1 , wherein said Arabidopsis ABPI endoplasmic reticulum target signal peptide is as set forth in SEQ ID NO: 4.
4. The nucleic acid construct of claim 1 , comprising a nucleic acid sequence encoding said endoplasmic reticulum targeting signal peptide as set forth in SEQ ID NO: 5.
5. The nucleic acid construct of claim 1 , wherein said endoplasmic reticulum retention signal peptide is as set forth in SEQ ID NO: 6.
6. The nucleic acid construct of claim 1 , comprising a nucleic acid sequence encoding said endoplasmic reticulum retention peptide as set forth in SEQ NO: 19.
7. The nucleic acid construct of claim 1 , wherein said nucleic acid sequence has a sequence as set forth in SEQ ID NO: 2.
8. The nucleic acid construct of claim 1 , wherein said human α-galactosidase protein has an amino acid sequence as set forth in SEQ ID NO: 1.
9. An isolated plant cell comprising the nucleic acid construct of claim 1 .
10. The cell of claim 9 , wherein said plant cell is a tobacco cell.
11. The cell of claim 10 , wherein said tobacco cell is a BY-2 cell.
12. A method of producing a catalytically active recombinant human α-galactosidase protein, comprising:
providing a cell according to claim 9 ; and
growing said cell so as to produce said catalytically active recombinant human α-galactosidase protein; and
isolating said catalytically active recombinant human α-galactosidase protein from said cell, wherein said catalytic activity is hydrolysis of p-nitrophenylalpha-D-galactopyranoside.
13. The method of claim 12 , wherein said cell is an isolated cell cultured in a cell culture medium.
14. The method of claim 13 , wherein said culturing is affected in a disposable bioreactor.
15. A human α-galactosidase protein having an N-terminal Glycine residue, wherein said human α-galactosidase protein is translationally fused at the C-terminal to an endoplasmic reticulum retention signal peptide and wherein said human α-galactosidase protein is catalytically active as determined by p-nitrophenylalpha-D-galactopyranoside assay.
16. The human α-galactosidase protein of claim 15 , having an amino acid sequence as set forth in SEQ ID NO: 16.
17. A pharmaceutical composition comprising, as an active ingredient, the human α-galactosidase protein of claim 15 and a pharmaceutically acceptable carrier.
18. A pharmaceutical composition comprising the human α-galactosidase protein of claim 15 , having a glycan structure comprising nine mannose residues, wherein three are exposed mannose residues, and a pharmaceutically acceptable carrier.
19. A pharmaceutical composition comprising, as an active ingredient, a population of human α-galactosidase proteins of claim 15 , wherein at least 0.5% of said population has a glycan structure comprising nine mannose residues, wherein three are exposed mannose residues.
20. A pharmaceutical composition comprising, as an active ingredient, a population of human α-galactosidase proteins of claim 15 , wherein the predominant glycan structures of said population of human α-galactosidase proteins are mannose 4-β-(1,2) xylose (M4X); mannose 3-β-(1,2) xylose-α-(1,3) fucose [Fc(3)M3X]; and mannose 8 (M8), and a pharmaceutically acceptable carrier.
21. A pharmaceutical composition comprising, as an active ingredient, the cell of claim 9 and a pharmaceutically acceptable carrier.
22. A method of treating Fabry's disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 17 .
23. A method of treating Fabry's disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 20 .
24. A method of treating Fabry's disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 21 .