Compositions of Aminoacyl-tRNA Synthetase and Uses Thereof

• US 20100167347A1
• Filed: 03/15/2010
• Published: 07/01/2010
• 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 7,858,344 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

1-18. -18. (canceled)

19. A cell comprising a translation system, wherein the translation system comprises an aminoacyl tRNA synthetase (RS) nucleic acid sequence selected from the group consisting of SEQ ID NO:

17, and a complementary polynucleotide sequence of SEQ ID NO;

17.

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. The cell of claim 19, wherein the cell is an E. coli.

31-35. -35. (canceled)

36. A vector comprising a polynucleotide encoding an RS having a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:

17, and a complementary polynucleotide sequence 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.

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

17, and a complementary polynucleotide sequence 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.

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

42-43. -43. (canceled)

45. A polynucleotide having a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:

17, and a complementary polynucleotide sequence thereof.