Compositions of aminoacyl-tRNA synthetase and uses thereof

• US 20090123971A1
• Filed: 12/01/2005
• Published: 05/14/2009
• Est. Priority Date: 12/22/2004
• Status: Active Grant

• Current Assignee: Ambrx, Inc. (Johnson & Johnson)
• Original Assignee: Ambrx, Inc. (Johnson & Johnson)
• Inventors: Paulsel, Andrew, Cho, Ho Sung
• Granted Patent: US 7,736,872 B2
• Time in Patent Office: Days
• US Class Current: 435/69.400
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione

Abstract

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNA'"'"'s, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNA'"'"'s/synthetases are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins using these orthogonal pairs.

Claims

1. A composition comprising an aminoacyl tRNA synthetase (RS) having the nucleic acid sequence selected from the group consisting of SEQ ID NO:

4, a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO;

5, a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO;

17, a complementary polynucleotide sequence thereof, and a conservative variation thereof.

2. The composition of claim 1, wherein said RS preferentially aminoacylates a tRNA.

3. The composition of claim 2, wherein said tRNA is aminoacylated with a non-naturally encoded amino acid.

4. The composition of claim 3, wherein said non-naturally encoded amino acid is para-acetylphenylalanine.

5. A composition comprising the RS of claim 1 and a tRNA, wherein said tRNA is chemically aminoacylated.

6. A composition comprising the RS of claim 1 and a tRNA, wherein said tRNA is enzymatically aminoacylated.

7. The composition of claim 6, wherein said tRNA is enzymatically aminoacylated by the RS.

8. The composition of claim 6, wherein said tRNA is enzymatically aminoacylated by a ribozyme.

9. The composition of claim 1, further comprising a tRNA, wherein the tRNA is aminoacylated with an amino acid by the RS.

10. The composition of claim 9, wherein said amino acid is a non-naturally encoded amino acid.

11. The composition of claim 10, wherein said non-naturally encoded amino acid is para-acetylphenylalanine.

12. The composition of claim 9, wherein the tRNA is derived from an archael tRNA.

13. The composition of claim 9, wherein the tRNA is derived from M janneschii.

14. The composition of claim 1, further comprising a translation system.

15. The composition of claim 14, wherein said translation system is a cell-free translation system.

16. The composition of claim 14, wherein said translation system is a cell lysate.

17. The composition of claim 14, wherein said translation system is a reconstituted system.

18. The composition of claim 14, wherein said translation system is a cellular translation system.

19. A cell comprising a translation system, wherein the translation system comprises the RS of claim 1.

20. The cell of claim 19, wherein the cell is a eukaryotic cell.

21. The cell of claim 20, wherein the eukaryotic cell is a yeast cell.

22. The cell of claim 20, wherein the eukaryotic cell is a fungal cell.

23. The cell of claim 20, wherein the eukaryotic cell is a mammalian cell.

24. The cell of claim 20, wherein the eukaryotic cell is an insect cell.

25. The cell of claim 20, wherein the eukaryotic cell is a plant cell.

26. The cell of claim 19, wherein the cell is a non-eukaryotic cell.

27. The cell of claim 26, wherein the non-eukaryotic cell is an E. coli cell.

28. The cell of claim 19, further comprising a tRNA that is aminoacylated by the RS and polynucleotide encoding a polypeptide of interest, wherein the polynucleotide comprises a selector codon that is recognized by the tRNA.

29. The cell of claim 28, wherein said polypeptide of interest is human growth hormone.

30. An E. coli cell comprising the RS of claim 1.

31. A yeast cell comprising the RS of claim 1.

32. A fungal cell comprising the RS of claim 1.

33. A mammalian cell comprising the RS of claim 1.

34. An insect cell comprising the RS of claim 1.

35. A plant cell comprising the RS of claim 1.

40. A method of producing a polypeptide in a cell with a selected amino acid at a specified position, the method comprising:

growing, in an appropriate medium, the cell, where the cell comprises a nucleic acid that comprises at least one selector codon and encodes a polypeptide; and

,

providing the selected amino acid;

wherein the cell further comprises;

an orthogonal RS(O—

RS) of claim 1 that functions in the cell wherein said RS has an amino acid sequence selected from the group consisting of SEQ ID NO;

5, SEQ ID NO;

17, a complementary polynucleotide sequence thereof, and a conservative variation thereof; and

,

an orthogonal tRNA (O-tRNA), that recognizes the selector codon wherein said O—

RS aminoacylates the O-tRNA with the selected amino acid.

41. The method of claim 40 wherein said selected amino acid is para-acetyl phenylalanine.

42. A polypeptide produced by the method of claim 40.

43. The polypeptide of claim 40 wherein said selected amino acid is para-acetyl phenylalanine.

44. The method of claim 40 wherein said polypeptide is human growth hormone.

36. A vector comprising a polynucleotide encoding an RS having the nucleic acid sequence selected from the group consisting of SEQ ID NO:

4, a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO;

5, a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO;

17, a complementary polynucleotide sequence thereof, and a conservative variation thereof.

37. The vector of claim 36, wherein the vector comprises a plasmid, a cosmid, a phage, or a virus.

38. The vector of claim 36, wherein the vector is an expression vector.

39. A cell comprising the vector of claim 36.

45. A polynucleotide having a nucleic acid sequence selected from a group consisting of SEQ ID No. 4, a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:

5, a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO;

17, a complementary polynucleotide sequence thereof, and a conservative variation thereof.