Compositions of aminoacyl-TRNA synthetase and uses thereof
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.
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.