Claims
- 1. An isolated human cystathionine β-synthase variant selected from the group consisting of:
a) a protein consisting essentially of an amino acid spanning from a starting position of one of amino acid residues from about 66-84 of SEQ ID NO: 2 to an ending position of one of amino acid residues from about 382-532 or 543-551 of SEQ ID NO: 2; and b) a homologue of the protein of (a), wherein said homologue consists essentially of an amino acid sequence that is at least about 70% identical to said amino acid sequence of (a); wherein said isolated human cystathionine β-synthase variant catalyzes the formation of cystathionine and does not bind heme.
- 2. The isolated human cystathionine β-synthase variant of claim 1, wherein said variant has specific activity that is at least about 0.5% of the specific activity of the wild-type human cystathionine β-synthase comprising SEQ ID NO: 2.
- 3. The isolated human cystathionine β-synthase variant of claim 1, wherein said variant has specific activity that is at least about 1% of the specific activity of the wild-type human cystathionine β-synthase comprising SEQ ID NO: 2.
- 4. The isolated human cystathionine β-synthase variant of claim 1, wherein said variant has specific activity that is at least about 10% of the specific activity of the wild-type human cystathionine β-synthase comprising SEQ ID NO: 2.
- 5. The isolated human cystathionine β-synthase variant of claim 1, wherein said variant has specific activity that is at least about 20% of the specific activity of the wild-type human cystathionine β-synthase comprising SEQ ID NO: 2.
- 6. The isolated human cystathionine β-synthase variant of claim 1, wherein said starting position is one of amino acid residues from about 66-71 of SEQ ID NO: 2.
- 7. The isolated human cystathionine β-synthase variant of claim 1, wherein said starting position is one of amino acid residues from about 70-84 of SEQ ID NO: 2.
- 8. The isolated human cystathionine β-synthase variant of claim 1, wherein said starting position is about position 70 or 71 of SEQ ID NO: 2.
- 9. The isolated human cystathionine β-synthase variant of claim 1, wherein said ending position is one of amino acid residues from about 382-523 or 543-551 of SEQ ID NO: 2.
- 10. The isolated human cystathionine β-synthase variant of claim 1, wherein said ending position is one of amino acid residues from about 400-523 or 543-551 of SEQ ID NO: 2.
- 11. The isolated human cystathionine β-synthase variant of claim 1, wherein said ending position is one of amino acid residues from about 413-523 or 543-551 of SEQ ID NO: 2.
- 12. The isolated human cystathionine β-synthase variant of claim 1, wherein said ending position is one of amino acid residues from about 441-523 or 543-551 of SEQ ID NO: 2.
- 13. The isolated human cystathionine β-synthase variant of claim 1, wherein said ending position is one of amino acid residues from about 488-523 or 543-551 of SEQ ID NO: 2.
- 14. The isolated human cystathionine β-synthase variant of claim 1, wherein said ending position is one of amino acid residues from about 496-523 or 543-551 of SEQ ID NO: 2.
- 15. The isolated human cystathionine β-synthase variant of claim 1, wherein said ending position is one of amino acid residues from about 543-551 of SEQ ID NO: 2.
- 16. The isolated human cystathionine β-synthase variant of claim 1, wherein said protein of (a) consists essentially of an amino acid spanning from a starting position of one of amino acid residues from about 66-84 of SEQ ID NO: 2 to an ending position of one of amino acid residues from about 400-523 or 543-551 of SEQ ID NO: 2.
- 17. The isolated human cystathionine β-synthase variant of claim 1, wherein said protein of (a) consists essentially of an amino acid spanning from a starting position of one of amino acid residues from about 70 or 71 of SEQ ID NO: 2 to an ending position of one of amino acid residues from about 400-523 or 543-551 of SEQ ID NO: 2.
- 18. The isolated human cystathionine β-synthase variant of claim 1, wherein said protein of (a) consists essentially of an amino acid spanning from a starting position of one of amino acid residues from about 66-84 of SEQ ID NO: 2 to an ending position of about 551 of SEQ ID NO: 2.
- 19. The isolated human cystathionine β-synthase variant of claim 1, wherein said protein of (a) consists essentially of an amino acid spanning from a starting position of one of amino acid residues from about 70 or 71 of SEQ ID NO: 2 to an ending position of about 400 of SEQ ID NO: 2.
- 20. The isolated human cystathionine β-synthase variant of claim 1, wherein said protein of (a) consists essentially of an amino acid spanning from a starting position of about position 70 or 71 of SEQ ID NO: 2 to an ending position of one of amino acid residues from about 544-551 of SEQ ID NO: 2.
- 21. The isolated human cystathionine β-synthase variant of claim 1, wherein said protein of (a) consists essentially of an amino acid spanning from a starting position of about position 70 or 71 of SEQ ID NO: 2 to an ending position of about 551 of SEQ ID NO: 2.
- 22. The isolated human cystathionine β-synthase variant of claim 1, wherein said homologue is at least about 80% identical to said amino acid sequence of (a).
- 23. The isolated human cystathionine β-synthase variant of claim 1, wherein said homologue is at least about 90% identical to said amino acid sequence of (a).
- 24. The isolated human cystathionine β-synthase variant of claim 1, wherein said variant comprises no more than one or two amino acid residues at the N-terminus that is not a residue of the naturally occurring human cystathionine β-synthase amino acid sequence.
- 25. An isolated fusion protein comprising the isolated human cystathionine β-synthase variant of claim 1 linked to a heterologous protein sequence.
- 26. A composition comprising the isolated human cystathionine β-synthase variant of claim 1.
- 27. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the human cystathionine β-synthase variant of claim 1.
- 28. A recombinant nucleic acid molecule comprising the nucleic acid sequence of claim 27, operatively linked to a expression control sequence.
- 29. A recombinant host cell that is transfected with and expresses the recombinant nucleic acid molecule of claim 28.
- 30. A method to treat homocystinuria, comprising administering to a patient the isolated protein of claim 1.
- 31. A method to treat homocystinuria, comprising administering to a patient the recombinant nucleic acid molecule of claim 28.
- 32. An isolated human cystathionine β-synthase variant consisting essentially of an amino acid sequence that differs from SEQ ID NO: 2 by at least a deletion or mutation of Cys52 and His65 of SEQ ID NO: 2, wherein said variant catalyzes the formation of cystathionine and does not bind heme.
- 33. The isolated human cystathionine β-synthase variant of claim 32, wherein said variant further differs from SEQ ID NO: 2 by a deletion of at least amino acid positions 1-39 of SEQ ID NO: 2.
- 34. The isolated human cystathionine β-synthase variant of claim 32, wherein said variant further differs from SEQ ID NO: 2 by a deletion of at least amino acid positions 1-50 of SEQ ID NO: 2.
- 35. The isolated human cystathionine β-synthase variant of claim 32, wherein said variant further differs from SEQ ID NO: 2 by a deletion of at least amino acid positions 1-60 of SEQ ID NO: 2.
- 36. The isolated human cystathionine β-synthase variant of claim 32, wherein said variant further differs from SEQ ID NO: 2 by a deletion of at least amino acid positions 1-70 of SEQ ID NO: 2.
- 37. The isolated human cystathionine β-synthase variant of claim 32, wherein said variant further differs from SEQ ID NO: 2 by a deletion of between about 1 and about 8 amino acids from the C-terminus of SEQ ID NO: 2.
- 38. The isolated human cystathionine β-synthase variant of claim 32, wherein said variant further differs from SEQ ID NO: 2 by a deletion of between about 19 and about 169 amino acids from the C-terminus of SEQ ID NO: 2.
- 39. The isolated human cystathionine β-synthase variant of claim 32, wherein said variant further differs from SEQ ID NO: 2 by a deletion of between about 28 and about 169 amino acids from the C-terminus of SEQ ID NO: 2.
- 40. The isolated human cystathionine β-synthase variant of claim 32, wherein said variant further differs from SEQ ID NO: 2 by a deletion of between about 28 and about 151 amino acids from the C-terminus of SEQ ID NO: 2.
- 41. The isolated human cystathionine β-synthase variant of claim 32, wherein said variant comprises no more than one or two amino acid residues at the N-terminus that is not a residue of the naturally occurring human cystathionine β-synthase amino acid sequence.
- 42. An isolated fusion protein comprising the isolated human cystathionine β-synthase variant of claim 41 linked to a heterologous protein sequence.
- 43. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the human cystathionine β-synthase variant of claim 32.
- 44. A recombinant nucleic acid molecule comprising the nucleic acid sequence of claim 43, operatively linked to a expression control sequence.
- 45. An isolated, recombinant human cystathionine β-synthase protein comprising no more than one or two amino acid residues at the N-terminus that is not a residue of the naturally occurring human cystathionine β-synthase amino acid sequence, wherein said human cystathionine β-synthase protein comprises an amino acid sequence selected from the group consisting of:
a) positions 2-551 of SEQ ID NO: 2; b) an amino acid sequence that is at least about 70% identical to positions 2-551 of SEQ ID NO: 2; and c) an enzymatically active fragment of SEQ ID NO: 2, wherein said fragment catalyzes the formation of cystathionine.
- 46. The isolated, recombinant human cystathionine β-synthase protein of claim 45, wherein the amino acid sequence further differs from positions 2-551 of SEQ ID NO: 2 by at least one deletion or mutation of an amino acid residue of SEQ ID NO: 2 selected from the group consisting of: Cys52 and His65 of SEQ ID NO: 2, wherein said variant catalyzes the formation of cystathionine and has a reduced ability to bind heme.
- 47. The isolated, recombinant human cystathionine β-synthase protein of claim 45, wherein the enzymatically active fragment differs from positions 2-551 of SEQ ID NO: 2 by a deletion of at least amino acid positions 2-39 of SEQ ID NO: 2.
- 48. The isolated, recombinant human cystathionine β-synthase protein of claim 45, wherein the enzymatically active fragment differs from positions 2-551 of SEQ ID NO: 2 by a deletion of at least amino acid positions 2-65 of SEQ ID NO: 2.
- 49. The isolated, recombinant human cystathionine β-synthase protein of claim 45, wherein the enzymatically active fragment differs from positions 2-551 of SEQ ID NO: 2 by a deletion of at least amino acid positions 2-70 or 2-71 of SEQ ID NO: 2.
- 50. The isolated, recombinant human cystathionine β-synthase protein of claim 45, wherein the enzymatically active fragment differs from positions 2-551 of SEQ ID NO: 2 by a deletion of at least amino acid positions 2-83 of SEQ ID NO: 2.
- 51. The isolated, recombinant human cystathionine β-synthase protein of claim 45, wherein the enzymatically active fragment differs from positions 2-551 of SEQ ID NO: 2 by a deletion of between about 1 and about 8 amino acids from the C-terminus of SEQ ID NO: 2.
- 52. The isolated, recombinant human cystathionine β-synthase protein of claim 45, wherein the enzymatically active fragment differs from positions 2-551 of SEQ ID NO: 2 by a deletion of between about 19 and about 169 amino acids from the C-terminus of SEQ ID NO: 2.
- 53. The isolated, recombinant human cystathionine β-synthase protein of claim 45, wherein the enzymatically active fragment differs from positions 2-551 of SEQ ID NO: 2 by a deletion of between about 28 and about 169 amino acids from the C-terminus of SEQ ID NO: 2.
- 54. The isolated, recombinant human cystathionine β-synthase protein of claim 45, wherein the enzymatically active fragment differs from positions 2-551 of SEQ ID NO: 2 by a deletion of between about 28 and about 151 amino acids from the C-terminus of SEQ ID NO: 2.
- 55. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the recombinant human cystathionine β-synthase of claim 45.
- 56. A recombinant nucleic acid molecule comprising the nucleic acid sequence of claim 55, operatively linked to a expression control sequence.
- 57. A method to produce a recombinant human cystathionine β-synthase comprising:
a) transfecting a recombinant host cell with a recombinant nucleic acid molecule comprising a first nucleic acid sequence encoding a human cystathionine β-synthase or homologue thereof having human cystathionine β-synthase biological activity, wherein the recombinant nucleic acid molecule comprises a recombinant expression vector operatively linked to said first nucleic acid sequence, said expression vector comprising:
i) a second nucleic acid sequence encoding a fusion segment which is linked to the N-terminus of said first nucleic acid sequence by a linker region that will produce a human cystathionine β-synthase fusion protein comprising said fusion segment when said recombinant nucleic acid molecule is expressed; and ii) a human rhinovirus 3C protease recognition sequence within said linker region; wherein the 5′ nucleotide of the nucleic acid sequence encoding the N-terminal amino acid residue of the human cystathionine β-synthase or homologue thereof is contiguous with the 3′ nucleotide of the nucleic acid sequence encoding the two amino acid residues that occur immediately C-terminal to the human rhinovirus 3C protease recognition site, such that a human cystathionine β-synthase expressed by said recombinant nucleic acid molecule will contain at its N-terminus the two C-terminal amino acid residues of the human rhinovirus 3C protease recognition site; b) culturing the transfected host cell from (a) under conditions effective to produce said recombinant human cystathionine β-synthase fusion protein; c) contacting the recombinant human cystathionine β-synthase fusion protein with a human rhinovirus 3C protease to cleave the fusion segment from the recombinant human cystathionine β-synthase; and d) recovering the recombinant human cystathionine β-synthase as a substantially purified recombinant protein.
- 58. The method of claim 57, wherein the nucleic acid sequence encoding a human cystathionine β-synthase encodes a wild-type human cystathionine β-synthase comprising positions 3-551 of SEQ ID NO: 2.
- 59. The method of claim 57, wherein the nucleic acid sequence encoding a human cystathionine β-synthase encodes a homologue of the wild type human cystathionine β-synthase that is at least about 70% identical to SEQ ID NO: 2.
- 60. The method of claim 57, wherein the nucleic acid sequence encoding a human cystathionine β-synthase encodes a truncated variant of the wild type human cystathionine β-synthase having enzymatic activity.
- 61. The method of claim 60, wherein said truncated variant does not bind heme.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. § 119(e) from U.S. Provisional Application Serial No. 60/389,541, filed Jun. 17, 2002. The entire disclosure of U.S. Provisional Application Serial No. 60/389,541 is incorporated herein by reference.
GOVERNMENT RIGHTS
[0002] This invention was supported in part with funding provided by NIH Grant Nos. P01HD0805 and HL65217, each awarded by the National Institutes of Health. The government may have certain rights to this invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60389541 |
Jun 2002 |
US |