In vivo incorporation of alkynyl amino acids into proteins in eubacteria
The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate alkynyl amino acids such as para-propargyloxyphenylalanine into proteins produced in a eubacteria host such as E. coli . The invention provides novel orthoghonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing alkynyl amino acids, and cellular translation systems.
1. A eubacterial cell comprising a first orthogonal aminoacyl-tRNA synthetase (O-RS) that functions in the cell, wherein the O-RS preferentially aminoacylates a first orthogonal tRNA (O-tRNA) with a first unnatural amino acid that is an alkynyl amino acid;
wherein said first O-RS is at least 90% identical to the wild-type Methanococcus jannaschii tyrosyl-tRNA synthetase having the amino acid sequence of SEQ ID NO: 2, wherein the O-RS has an amino acid sequence comprising:
(a) alanine at amino acid position 32;
(b) alanine or Leu at amino acid position 158; and
(c) alanine or proline at amino acid position 162; and,
wherein the alkynyl amino acid is a para-substituted tyrosine or a para-substituted phenylalanine.
2. The cell of claim 1 , wherein the eubacterial cell is an E. coli cell.
3. The cell of claim 1 , wherein the O-RS is derived from a Methanococcus jannaschii aminoacyl-tRNA synthetase.
4. The cell of claim 1 , wherein the O-RS is derived from a Methanococcus jannaschii tyrosyl-tRNA synthetase.
5. The cell of claim 1 , wherein the O-RS is derived from the wild-type Methanococcus jannaschii tyrosyl-tRNA synthetase having the amino acid sequence of SEQ ID NO: 2.
6. The cell of claim 1 , wherein the O-RS comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, and 18.
7. The cell of claim 1 , wherein the cell comprises a polynucleotide encoding the O-RS, wherein the O-RS comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, and 18.
8. The cell of claim 7 , wherein the polynucleotide is selected from the nucleotide sequences of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17 or 19.
9. The cell of claim 1 , wherein the O-tRNA is an amber suppressor tRNA.
10. The cell of claim 1 , wherein the O-tRNA comprises or is encoded by a polynucleotide sequence set forth in SEQ ID NO: 1.
11. The cell of claim 1 , wherein said alkynyl amino acid is para -propargyloxyphenylalanine.
12. The cell of claim 1 , comprising a nucleic acid comprising at least one selector codon, wherein said selector codon is recognized by said first O-tRNA.
13. The cell of claim 12 , comprising a second O-RS and a second O-tRNA, wherein the second O-RS preferentially aminoacylates the second O-tRNA with a second unnatural amino acid that is different from the first unnatural amino acid, and wherein the second O-tRNA recognizes a selector codon that is different from the selector codon recognized by the first O-tRNA.
14. The cell of claim 1 , comprising said alkynyl amino acid.
15. The cell of claim 14 , wherein said alkynyl amino acid is para-propargyloxyphenylalanine.
16. The cell of claim 1 , comprising a translation system.
17. The cell of claim 16 , said translation system comprising:
(a) said O-RS;
(b) said O-tRNA;
(c) a nucleic acid encoding a polypeptide of interest, the nucleic acid comprising at least one selector codon, wherein the selector codon is recognized by said O-tRNA; and,
(d) an alkynyl amino acid, wherein said O-RS is capable of charging said O-tRNA with said alkynyl amino acid.