Human cystathionine β-synthase variants and methods of production thereof
View Patent ↗Human cystathionine β-synthase variants are disclosed, as well as a method to produce recombinant human cystathionine β-synthase and variants thereof. More particularly, the role of both the N-terminal and C-terminal regions of human CBS has been studied, and a variety of truncation mutants and modified CBS homologues are described. In addition, a method to express and purify recombinant human cystathionine β-synthase (CBS) and variants thereof which have only one or two additional amino acid residues at the N-terminus are described.
1. A fusion protein comprising a truncated form of human cystathionine-β-synthase (CBS) protein having human cystathionine-β-synthase biological activity, said protein having an amino acid sequence that is at least 95% identical to amino acid sequence spanning from a starting position of amino acid residue 1 or one of amino acid residues from 66-84 of SEQ ID NO:2 to an ending position of one of amino acid residues from 382-532, 382-550 or 543-550 of SEQ ID NO:2.
2. The fusion protein of claim 1 , wherein said truncated form of human cystathionine-β-synthase protein has an amino acid sequence that spans from a starting position of amino acid residue 1 of SEQ ID NO:2.
3. The fusion protein of claim 1 , wherein said truncated form of human cystathionine-β-synthase protein has an amino acid sequence that spans from a starting position of one of amino acid residues from 66-84 of SEQ ID NO:2.
4. The fusion protein of claim 1 , wherein said truncated form of human cystathionine-β-synthase protein has an amino acid sequence that spans from a starting position of one of amino acid residues from 66-71 of SEQ ID NO:2.
5. The fusion protein of claim 1 , wherein said truncated form of human cystathionine-β-synthase protein has an amino acid sequence that spans from a starting position of one of amino acid residues from 70-84 of SEQ ID NO:2.
6. The fusion protein of claim 1 , wherein said truncated form of human cystathionine-β-synthase protein has an amino acid sequence that spans from a starting position of one of amino acid residues from 70 or 71 of SEQ ID NO:2.
7. The fusion protein of claim 1 , wherein said truncated form of human cystathionine-β-synthase protein has an amino acid sequence that ends at a position of one of amino acid residues from 382-532.
8. The fusion protein of claim 1 , wherein said truncated form of human cystathionine-β-synthase protein has an amino acid sequence that ends at a position of one of amino acid residues from 382-550.
9. The fusion protein of claim 1 , wherein said truncated form of human cystathionine-β-synthase protein has an amino acid sequence that ends at a position of one of amino acid residues from 543-550.
10. The fusion protein of claim 1 , wherein said truncated form of human cystathionine-β-synthase protein has an amino acid sequence that ends at a position of one of amino acid residues from 400-523.
11. The fusion protein of claim 1 , wherein said truncated form of human cystathionine-β-synthase protein has an amino acid sequence that ends at a position of one of amino acid residues from 488-523.
12. The fusion protein of claim 1 , wherein said truncated form of human cystathionine-β-synthase protein has an amino acid sequence that ends at a position of one of amino acid residues from 496-523.
13. The fusion protein of claim 1 , wherein said truncated form of human cystathionine-β-synthase protein does not bind heme.
14. The fusion protein of claim 1 , wherein said truncated form of human cystathionine-β-synthase protein contains at its N-terminus the two C-terminal amino acid residues of the rhinovirus 3C protease recognition site.
15. The fusion protein of claim 1 , wherein said fusion protein spans from a starting position of amino acid residue 1, or a starting position of one of amino acid residues from 66-84, or a starting position of one of amino acid residues from 66-71, or a starting position of one of amino acid residues from 70-71, or a starting position of one of amino acid residues from 70-84, and wherein said fusion protein has an amino acid sequence that ends at a position of one of amino acid residues from 382-532, or ends at a position of one of amino acid residues from 382-550, or ends at a position of one of amino acid residues from 400-523, or ends at a position of one of amino acid residues from 488-523, or ends at a position of one of amino acid residues from 496-523, and further comprises glutationine-S-transferase, a polymer of histidine comprising from 3 to 9 histidine residues, or a streptavidin tag comprising the sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys.
16. The fusion protein of claim 15 , wherein glutationine-S-transferase, a polymer of histidine comprising from 3 to 9 histidine residues, or a streptavidin tag comprising the sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys is covalently linked at the carboxyl terminus of the truncated form of human cystathionine-β-synthase protein.
17. The fusion protein of claim 15 , wherein glutationine-S-transferase, a polymer of histidine comprising from 3 to 9 histidine residues, or a streptavidin tag comprising the sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys is covalently linked at the amino terminus of the truncated form of human cystathionine-β-synthase protein.
18. A method for treating homocystinuria, comprising administering to a patient a fusion protein according to claim 15 .
19. A composition comprising the fusion protein of claim 1 , and a pharmaceutically acceptable carrier.
20. A composition comprising the fusion protein of claim 15 , and a pharmaceutically acceptable carrier.