Claims
- 1. A method of generating tRNA molecules with noncognate amino acids comprising the steps of:
mutating one or more amino acids of a leucyl tRNA synthetase to alter activity of the leucyl tRNA synthetase amino acid editing mechanism; and aminoacylating a tRNAleu in the presence of a noncognate amino acid.
- 2. The method of claim 1, wherein a mutation is in the amino acid editing active site of leucyl tRNA synthetase, wherein the mutation alters leucyl tRNA synthetase activity.
- 3. The method of claim 1, wherein the amino acid is a threonine at position 252 of E. coli leucyl-tRNA synthetase.
- 4. The method of claim 3, wherein the threonine at position 252 is mutated to a phenylalanine.
- 5. The method of claim 3, wherein the threonine at position 252 is mutated to a tyrosine.
- 6. The method of claim 3, wherein the threonine at position 252 is mutated to an alanine.
- 7. The method of claim 3, wherein the threonine at position 252 is mutated to a tryptophan.
- 8. The method of claim 1, wherein the amino acid is an aspartic acid at position 419 of S. cerevisiae leucyl-tRNA synthetase.
- 9. The method of claim 8, wherein the aspartic acid at position 419 is mutated to an alanine.
- 10. The method of claim 1, wherein the amino acid is an aspartic acid at position 345 of E. coli leucyl-tRNA synthetase
- 11. The method of claim 10, wherein the aspartic acid at position 345 is mutated to an alanine.
- 12. The method of claim 1, wherein the aspartic acids at position 342 and 345 of E. coli leucyl-tRNA synthetase are mutated to an alanine.
- 13. The method of claim 1, wherein the threonine at position 252 is mutated to another amino acid and an aspartic acid at position 342 of E. coli leucyl-tRNA synthetase is mutated to alanine.
- 14. The method of claim 1, wherein the threonine at position 252 is mutated to another amino acid and an aspartic acid at position 345 of E. coli leucyl-tRNA synthetase is mutated to an alanine.
- 15. The method of claim 1, wherein the threonine at position 252 is mutated to an amino acid from the group consisting of alanine, tyrosine, and phenylalanine and an aspartic acid at position 345 of E. coli leucyl-tRNA synthetase is mutated to any amino acid.
- 16. The method of claim 1, wherein the threonine at position 252 is mutated to to an amino acid from the group consisting of alanine, tyrosine, and phenylalanine and an aspartic acid at position 342 of E. coli leucyl-tRNA synthetase is mutated to any amino acid.
- 17. The method of claim 1, wherein the threonine at position 252, the aspartic acid at position 342 and the aspartic acid at position 345 of E. coli leucyl-tRNA synthetase are mutated to alanine.
- 18. The method of claim 1, wherein the threonine at position 252, the aspartic acid at position 342 and the aspartic acid at position 345 of E. coli leucyl-tRNA synthetase are mutated to any amino acid.
- 19. The method of claim 1, wherein leucyl tRNA synthetase is from the group consisting of a derivative, natural, synthetic analog, engineered enzyme, mimetic, single mutant, multiple mutant, mutant with deletions, chimeric molecule, versions that are cognate to other amino acids, and combinations thereof and from any eukaryotic, bacterial, and archaebacterial organism.
- 20. The method of claim 1, wherein the altered activity of the leucyl tRNA synthetase from the group consisting of reduced activity and increased activity.
- 21. A method of generating tRNA synthetase molecules exhibiting altered specificity for a cognate amino acid comprising the step of mutating one of more amino acids of the leucyl tRNA synthetase to alter the specificity for amino acid activation and aminoacylation of the cognate amino acid.
- 22. The method of claim 21, wherein the amino acid is a tyrosine at position 330 of E. coli leucyl-tRNA synthetase
- 23. The method of claim 21, wherein the amino acid is mutated to alanine.
- 24. The method of claim 21, wherein the amino acid is methionine at position 40 of E. coli leucyl-tRNA synthetase.
- 25. The method of claim 21, wherein the amino acid is mutated to a serine.
- 26. The method of claim 21, wherein leucyl tRNA synthetase is from the group consisting of a derivative, natural, synthetic analog, engineered enzyme, mimetic, single mutant, multiple mutant, mutant with deletions, chimeric molecule, versions that are cognate to other amino acids, and combinations thereof and from any eukaryotic, bacterial, and archaebacterial organism.
- 27. The method of claim 21, wherein the altered activity of the leucyl tRNA synthetase is an increased activity that increases aminoacylation.
- 28. A method of generating leucyl-tRNA synthetase molecules with altered specificity for non-leucine amino acids comprising the steps of:
mutating one of more amino acids of the leucyl tRNA synthetase to alter the specificity for amino acid activation and aminoacylation of the leucyl tRNA synthetase to a non-leucine amino acid; and aminoacylating a tRNAleu with a leucyl tRNA synthetase and a non-leucine amino acid.
- 29. The method of claim 28, wherein leucyl tRNA synthetase is from the group consisting of a derivative, natural, synthetic analog, engineered enzyme, mimetic, single mutant, multiple mutant, mutant with deletions, chimeric molecule, versions that are cognate to other amino acids, and combinations thereof and from any eukaryotic, bacterial, and archaebacterial organism.
- 30. The method of claim 28, wherein the altered activity of the leucyl tRNA synthetase is an increased activity.
- 31. A method of generating a tRNA molecule with a noncognate amino acid comprising the steps of:
mutating an amino acid at position 238 within the amino acid binding pocket of the valyl tRNA synthetase to alter the valyl tRNA synthetase editing mechanism; and aminoacylating a tRNAval with the valyl tRNA synthetase and a noncognate amino acid.
- 32. The method of claim 31, wherein the aspartic acid at position 238 of E. coli is mutated to an alanine.
- 33. The method of claim 31, wherein to alter the valyl tRNA synthetase editing mechanism is to inactivate the valyl tRNA synthetase editing mechanism.
- 34. A method of linking covalently a non-leucine amino acid to a tRNAleu molecule comprising the steps of:
mutating one or more amino acids of the leucyl tRNA synthetase to alter the leucyl tRNA synthetase editing mechanism; and aminoacylating a tRNAleu molecule with a composition comprising the leucyl tRNA synthetase and a non-leucine amino acid.
- 35. The method of claim 34, wherein leucyl tRNA synthetase is from the group consisting of a derivative, natural, synthetic analog, engineered enzyme, mimetic, single mutant, multiple mutant, mutant with deletions, chimeric molecule, versions that are cognate to other amino acids, and combinations thereof and from any eukaryotic, bacterial, and archaebacterial organism.
- 36. An isolated polypeptide comprising the amino acid sequence of a mutant leucyl tRNA synthetase as set forth in TABLE 1, wherein the mutant leucyl tRNA synthetase is a mutant derived is from the group consisting of a derivative, natural, synthetic analog, engineered enzyme, mimetic, single mutant, multiple mutant, mutant with deletions, chimeric molecule, versions that are cognate to other amino acids, and combinations thereof and from any eukaryotic, bacterial, and archaebacterial organism.
- 37. An isolated nucleotide sequence encoding a polypeptide comprising the amino acid sequence of an E. coli mutant leucyl tRNA synthetase as set forth in TABLE 1.
- 38. An isolated nucleotide sequence encoding a polypeptide with a mutated editing comprising the amino acid sequence of leucyl tRNA synthetase is derived is from the group consisting of a derivative, natural, synthetic analog, engineered enzyme, mimetic, single mutant, multiple mutant, mutant with deletions, chimeric molecule, versions that are cognate to other amino acids, and combinations thereof and from any eukaryotic, bacterial, and archaebacterial organism.
- 39. An isolated polypeptide comprising the amino acid sequence of an E. coli mutant leucyl tRNA synthetase T252AID345A.
- 40. An isolated nucleotide sequence encoding a polypeptide comprising the amino acid sequence of an E. coli mutant leucyl tRNA synthetase T252A/D345A.
- 41. An isolated polypeptide comprising the amino acid sequence of an E. coli mutant leucyl tRNA synthetase D342A/D345A.
- 42. An isolated nucleotide sequence encoding a polypeptide comprising the amino acid sequence of an E. coli mutant leucyl tRNA synthetase D342A/D345A.
- 43. A method of producing a protein product with non-standard amino acids by comprising the steps of:
mutating an amino acid binding pocket at the editing site of the leucyl tRNA synthetase to alter its editing mechanism; admixing tRNA synthetases with a non-standard amino acid with a leucyl tRNA; and adding the leucyl tRNA with the non-cognate amino acid with a ribosome and ATP.
- 44. A method of producing a protein product with a non-non-cognate amino acid comprising the steps of:
mutating an amino acid binding pocket at the editing site of the leucyl tRNA synthetase to alter its editing mechanism; admixing tRNA synthetases with a non-cognate amino acid with a leucyl tRNA; and adding the leucyl tRNA with the non-cognate amino acid with a ribosome and ATP.
- 45. The method of claim 44, wherein two amino acids are mutated in the editing pocket of the active editing site.
- 46. The method of claim 44, wherein the three amino acids are mutated in the editing pocket of the active editing site.
- 47. The method of claim 44, wherein the four amino acids are mutated in the editing pocket of the active editing site.
- 48. The method of claim 44, wherein the amino acid that are mutated are an aspartic acid at position 342 and an aspartic acid at position 345.
- 49. The method of claim 44, wherein the two amino acids are mutated to alanine.
- 50. The method of claim 44, wherein leucyl tRNA synthetase is derived from the group consisting of a natural, synthetic analog, engineered enzyme, mimetic, mutant, and combinations thereof of leucyl tRNA synthetase from any eukaryotic, bacterial, and archaebacterial organism.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Application Serial No. 60/323,582 filed Sep. 20, 2001. The specification of the prior application is incorporated by reference into this specification.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] The present application was supported in part by the National Institutes of Health grant number GM63789. The government may have certain rights in the invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60323582 |
Sep 2001 |
US |