Compositions of aminoacyl-tRNA synthetase and uses thereof

• US 7,858,344 B2
• Filed: 03/15/2010
• Issued: 12/28/2010
• 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
• Primary Examiner(s): Rao; Manjunath N
• Assistant Examiner(s): Gebreyesus; Kagnew H
• Application Number:
  US12/724,365
• Publication Number:
  US 20100167347A1
• Time in Patent Office: 288 Days
• Field of Search: None
• US Class Current: 435/69.4
• 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. An isolated cell comprising a translation system where the translation system comprises a DNA sequence encoding the aminoacyl tRNA synthetase (RS) of SEQ ID NO:

17.

2. The cell of claim 1, wherein the cell is a eukaryotic cell.

3. The cell of claim 2, wherein the eukaryotic cell is a yeast cell.

4. The cell of claim 2, wherein the eukaryotic cell is a fungal cell.

5. The cell of claim 2, wherein the eukaryotic cell is a mammalian cell.

6. The cell of claim 2, wherein the eukaryotic cell is an insect cell.

7. The cell of claim 2, wherein the eukaryotic cell is a plant cell.

8. The cell of claim 1, wherein the cell is a non-eukaryotic cell.

9. The cell of claim 8, wherein the non-eukaryotic cell is an E. coli cell.

10. The cell of claim 1, further comprising a tRNA that is aminoacylated by said RS and a polynucleotide encoding a polypeptide of interest, wherein the polynucleotide comprises a selector codon that is recognized by the tRNA.

11. The cell of claim 10, wherein said polypeptide of interest is human growth hormone.

12. A vector comprising a DNA sequence encoding the RS of SEQ ID NO:

17.

13. The vector of claim 12, wherein the vector comprises a plasmid, a cosmid, a phage, or a virus.

14. The vector of claim 12, wherein the vector is an expression vector.

15. A cell comprising the vector of claim 12.

16. 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;

a DNA sequence that encodes an orthogonal RS (O—

RS) that functions in the cell wherein said RS has the amino acid sequence of SEQ ID NO;

17, and,

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

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

17. The method of claim 16 wherein said selected amino acid is para-acetyl phenylalanine.

18. The method of claim 16 wherein said polypeptide is human growth hormone.

19. An isolated polynucleotide encoding the amino acid sequence of SEQ ID NO:

17, and the complementary polynucleotide sequence thereof.