Compositions of aminoacyl-TRNA synthetase and uses thereof

• US 7,736,872 B2
• Filed: 12/01/2005
• Issued: 06/15/2010
• 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:
  US11/292,903
• Publication Number:
  US 20090123971A1
• Time in Patent Office: 1,657 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. A cell comprising a translation system, wherein the translation system comprises an aminoacyl tRNA synthetase (RS) encoded by the nucleic acid sequence consisting of SEQ ID NO:

4 and the complementary polynucleotide sequence of SEQ ID NO;

4.

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. The cell of claim 1, wherein the cell is an E. coli cell.

13. A vector comprising a polynucleotide sequence encoding an RS where the polynucleotide sequence consists of SEQ ID NO:

4, and the complementary polynucleotide sequence thereof.

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

15. The vector of claim 13, wherein the vector is an expression vector.

16. A cell comprising the vector of claim 13.

17. 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 O-RS is encoded by a polynucleotide sequence consisting of SEQ ID NO;

4 and the 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.

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

19. The method of claim 17 wherein said polypeptide is human growth hormone.

20. An isolated polynucleotide consisting of the nucleic acid sequence SEQ ID NO:

4, and the complementary polynucleotide sequence thereof.