Compositions of tRNA and uses thereof

• US 7,632,823 B2
• Filed: 12/01/2005
• Issued: 12/15/2009
• Est. Priority Date: 08/18/2005
• Status: Active Grant

• Current Assignee: Ambrx, Inc. (Johnson & Johnson)
• Original Assignee: Ambrx, Inc. (Johnson & Johnson)
• Inventors: Tian, Feng
• Primary Examiner(s): Wang; Andrew
• Assistant Examiner(s): Gebreyesus; Kagnew H
• Application Number:
  US11/293,629
• Publication Number:
  US 20080153745A1
• Time in Patent Office: 1,475 Days
• Field of Search: None
• US Class Current: 514/44.00R
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• 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 a tRNA having the nucleic acid sequence selected from the group consisting of SEQ ID NOs:

1, 2, and 3, and the full length complementary polynucleotide sequence that encodes SEQ ID NO;

1, 2 and 3 in a buffer.

2. The composition of claim 1, wherein said tRNA is aminoacylated.

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. The composition of claim 2, wherein said tRNA is chemically aminoacylated.

6. The composition of claim 2, wherein said tRNA is enzymatically aminoacylated.

7. The composition of claim 6, wherein said tRNA is enzymatically aminoacylated by an aminoacyl tRNA synthetase.

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

9. The composition of claim 1, further comprising an aminoacyl tRNA synthetase, wherein the tRNA is aminoacylated with an amino acid.

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 1, wherein the tRNA is derived from an archael tRNA.

13. The composition of claim 1, 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 tRNA 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 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 tRNA of claim 1.

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

32. A fungal cell comprising a tRNA of claim 1.

33. A mammalian cell comprising a tRNA of claim 1.

34. An insect cell comprising a tRNA of claim 1.

35. A plant cell comprising a tRNA of claim 1.

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

1, 2, and 3, and the full-length complementary polynucleotide sequence that encodes SEQ ID NO;

1, 2 and 3.

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.

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 tRNA (O-tRNA) that functions in the cell and recognizes the selector codon having the amino acid sequence selected from the group consisting of SEQ ID NO;

1, 2, 3, and the full-length complementary polynucleotide sequence that encodes SEQ ID NO;

1, 2 and 3; and

, an orthogonal aminoacyl-tRNA synthetase (O-RS), 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. The method of claim 40 wherein said polypeptide is human growth hormone.

43. A polynucleotide having a nucleic acid sequence selected from a group consisting of SEQ ID Nos. 1, 2, and 3, and the full-length complementary polynucleotide sequence thereof.