Suppressor tRNA transcription in vertebrate cells

• US 8,420,792 B2
• Filed: 09/07/2007
• Issued: 04/16/2013
• Est. Priority Date: 09/08/2006
• Status: Active Grant

• Current Assignee: Ambrx, Inc. (Johnson & Johnson)
• Original Assignee: Ambrx, Inc. (Johnson & Johnson)
• Inventors: Tian, Feng, Norman, Thea, Chu, Stephanie
• Primary Examiner(s): Joike, Michele K
• Application Number:
  US12/440,013
• Publication Number:
  US 20100159585A1
• Time in Patent Office: 2,048 Days
• Field of Search: None
• US Class Current: 536/23.1
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/85 for animal cells
• 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

This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in vertebrate cells. The components include orthogonal tRNA'"'"'s, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNA'"'"'s/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in vertebrate cells are also provided.

Claims

1. A tRNA transcription cassette wherein said cassette comprises nucleotides encoding a human tRNA and nucleotides encoding one or more Escherichia coli or Bacillus stearothermophilus tRNA.

2. The tRNA transcription cassette from claim 1, wherein said cassette also comprises a pol III promoter.

12. A vertebrate cell or cell line comprising the tRNA transcription cassette of claim 1.

13. The cell or cell line of claim 12, wherein the Escherichia coli or Bacillus stearothermophilus tRNA in the transcription cassette is aminoacylated with a non-natural amino acid.

20. The cell or cell line of claim 13, wherein the non-natural amino acid is selected from the group consisting of:

a p-acetyl-L-phenylalanine;

a p-iodo-L-phenylalanine;

an O-methyl-L-tyrosine;

a p-propargyloxyphenylalanine;

an L-3-(2-naphthyl)alanine;

a 3-methyl-phenylalanine;

an O-4-allyl-L-tyrosine;

a 4-propyl-L-tyrosine;

a tri-O-acetyl-GlcNAcβ

-serine;

an L-Dopa;

a fluorinated phenylalanine;

an isopropyl-L-phenylalanine;

a p-azido-L-phenylalanine;

a p-acyl-L-phenylalanine;

a p-benzoyl-L-phenylalanine;

an L-phosphoserine, a phosphonoserine;

a phosphonotyrosine;

a p-bromophenylalanine;

a p-amino-L-phenylalanine;

an isopropyl-L-phenylalanine;

an unnatural analogue of a tyrosine amino acid;

an unnatural analogue of a glutamine amino acid;

an unnatural analogue of a phenylalanine amino acid;

an unnatural analogue of a serine amino acid;

an unnatural analogue of a threonine amino acid;

an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or any combination thereof;

an amino acid with a photoactivatable cross-linker;

a spin-labeled amino acid;

a fluorescent amino acid;

a metal binding amino acid;

a metal-containing amino acid;

a radioactive amino acid;

a photocaged and/or photoisomerizable amino acid;

a biotin or biotin-analogue containing amino acid;

a keto containing amino acid;

an amino acid comprising polyethylene glycol or polyether;

a heavy atom substituted amino acid;

a chemically cleavable or photocleavable amino acid;

an amino acid with an elongated side chain;

an amino acid containing a toxic group;

a sugar substituted amino acid;

a carbon-linked sugar-containing amino acid;

a redox-active amino acid;

an α

-hydroxy containing acid;

an amino thio acid;

an α

,α

disubstituted amino acid;

a β

-amino acid;

a cyclic amino acid other than proline or histidine; and

an aromatic amino acid other than phenylalanine, tyrosine or tryptophan.

24. The cell or cell line of claim 13, wherein the tRNA recognizes a selector codon.

25. The cell or cell line of claim 24, wherein the selector codon is selected from the group consisting of:

amber codon, ochre codon, opal codon, or four or more base codons.

32. The cell or cell line of claim 13, further comprising a nucleic acid that comprises a polynucleotide that encodes a polypeptide of interest, wherein the polynucleotide comprises a selector codon that is recognized by the tRNA.

33. The cell or cell line of claim 32, wherein the polypeptide of interest is a therapeutic protein, a diagnostic protein, or an industrial enzyme.

34. The cell or cell line of claim 32, wherein the polypeptide of interest comprises a protein selected from the group consisting of:

a cytokine, a growth factor, a growth factor receptor, an interferon, an interleukin, an inflammatory molecule, an oncogene product, a peptide hormone, a signal transduction molecule, a steroid hormone receptor, erythropoietin (EPO), insulin, human growth hormone, an Alpha-1 antitrypsin, an Angiostatin, an Antihemolytic factor, an antibody, an Apolipoprotein, an Apoprotein, an Atrial natriuretic factor, an Atrial natriuretic polypeptide, an Atrial peptide, a C-X-C chemokine, T39765, NAP-2, ENA-78, a Gro-a, a Gro-b, a Gro-c, an IP-10, a GCP-2, an NAP-4, an SDF-1, a PF4, a MIG, a Calcitonin, a c-kit ligand, a CC chemokine, a Monocyte chemoattractant protein-1, a Monocyte chemoattractant protein-2, a Monocyte chemoattractant protein-3, a Monocyte inflammatory protein-1 alpha, a Monocyte inflammatory protein-1 beta, RANTES, I309, R83915, R91733, HCC1, T58847, D31065, T64262, a CD40, a CD40 ligand, a C-kit Ligand, a Collagen, a Colony stimulating factor (CSF), a Complement factor 5a, a Complement inhibitor, a Complement receptor 1, DHFR, an epithelial Neutrophil Activating Peptide-78, a GROα

/MGSA, a GROβ

, a GROγ

a MIP-1α

, a MIP-1δ

, a MCP-1, an Epidermal Growth Factor (EGF), an epithelial Neutrophil Activating Peptide, an Exfoliating toxin, a Factor IX, a Factor VII, a Factor VIII, a Factor X, a Fibroblast Growth Factor (FGF), a Fibrinogen, a Fibronectin, a G-CSF, a GM-CSF, a Glucocerebrosidase, a Gonadotropin, a Hedgehog protein, a Hemoglobin, a Hepatocyte Growth Factor (HGF), a Hirudin, a Human serum albumin, an ICAM-1, an ICAM-1 receptor, an LFA-1, an LFA-1 receptor, an Insulin-like Growth Factor (IGF), an IGF-I, an IGF-II, an IFN-α

, an IFN-β

, an IFN-γ

, an IL-1, an IL-2, an IL-3, an IL-4, an IL-5, an IL-6, an IL-7, an IL-8, an IL-9, an IL-10, an IL-11, an IL-12, a Keratinocyte Growth Factor (KGF), a Lactoferrin, a leukemia inhibitory factor, a Luciferase, a Neurturin, a Neutrophil inhibitory factor (NIF), an oncostatin M, an Osteogenic protein, a Parathyroid hormone, a PD-ECSF, a PDGF, a Pleiotropin, a Protein A, a Protein G, a Pyrogenic exotoxins A, B, or C, a Relaxin, a Renin, an SCF, a Soluble complement receptor I, a Soluble I-CAM 1, a Soluble interleukin receptor, a Soluble TNF receptor, a Somatomedin, a Somatostatin, a Somatotropin, a Streptokinase, a Superantigen, a Staphylococcal enterotoxins, an SEA, an SEB, an SEC1, an SEC2, an SEC3, an SED, an SEE, a Superoxide dismutase (SOD), a Toxic shock syndrome toxin, a Thymosin alpha 1, a Tissue plasminogen activator, a tumor growth factor (TGF), a TGF-α

, a TGF-β

, a Tumor Necrosis Factor, a Tumor Necrosis Factor alpha, a Tumor necrosis factor beta, a Tumor necrosis factor receptor (TNFR), a VLA-4 protein, a VCAM-1 protein, a Vascular Endothelial Growth Factor (VEGEF), a Urokinase, a Mos, a Ras, a Raf, a Met;

a p53, a Tat, a Fos, a Myc, a Jun, a Myb, a Rel, an estrogen receptor, a progesterone receptor, a testosterone receptor, an aldosterone receptor, an LDL receptor, a SCF/c-Kit, a CD40L/CD40, a VLA-4/VCAM-1, an ICAM-1/LFA-1, a hyalurin/CD44, and a corticosterone.

44. A kit for producing a protein that comprises at least one unnatural amino acid, the kit comprising:

a container containing the tRNA transcription cassette of claim 1.

45. The kit of claim 44, wherein the kit further comprises at least one unnatural amino acid.

46. The kit of claim 45, wherein the kit further comprises instructional materials for producing the protein.

3. A tRNA transcription cassette comprising 5′

flanking sequences, nucleotides encoding a human tRNA and nucleotides encoding one or more Escherichia coli or Bacillus stearothermaphilus tRNA.

4. The tRNA transcription cassette from claim 3, wherein the tRNA transcription cassette also comprises a pol III promoter.

5. The tRNA transcription cassette from claim 4, wherein the pol III promoter is H1 or U6.

14. A vertebrate cell or cell line comprising the tRNA transcription cassette of claim 3.

15. The cell or cell line of claim 14, wherein the Escherichia coli or Bacillus stearothermophilus tRNA in the transcription cassette is aminoacylated with a non-natural amino acid.

21. The cell or cell line of claim 15, wherein the non-natural amino acid is selected from the group consisting of:

a p-acetyl-L-phenylalanine;

a p-iodo-L-phenylalanine;

an O-methyl-L-tyrosine;

a p-propargyloxyphenylalanine;

an L-3-(2-naphthyl)alanine;

a 3-methyl-phenylalanine;

an O-4-allyl-L-tyrosine;

a 4-propyl-L-tyrosine;

a tri-O-acetyl-GlcNAcβ

-serine;

an L-Dopa;

a fluorinated phenylalanine;

an isopropyl-L-phenylalanine;

a p-azido-L-phenylalanine;

a p-acyl-L-phenylalanine;

a p-benzoyl-L-phenylalanine;

an L-phosphoserine;

a phosphonoserine;

a phosphonotyrosine;

a p-bromophenylalanine;

a p-amino-L-phenylalanine;

an isopropyl-L-phenylalanine;

an unnatural analogue of a tyrosine amino acid;

an unnatural analogue of a glutamine amino acid;

an unnatural analogue of a phenylalanine amino acid;

an unnatural analogue of a serine amino acid;

an unnatural analogue of a threonine amino acid;

an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or any combination thereof;

an amino acid with a photoactivatable cross-linker;

a spin-labeled amino acid;

a fluorescent amino acid;

a metal binding amino acid;

a metal-containing amino acid;

a radioactive amino acid;

a photocaged and/or photoisomerizable amino acid;

a biotin or biotin-analogue containing amino acid;

a keto containing amino acid;

an amino acid comprising polyethylene glycol or polyether;

a heavy atom substituted amino acid;

a chemically cleavable or photocleavable amino acid;

an amino acid with an elongated side chain;

an amino acid containing a toxic group;

a sugar substituted amino acid;

a carbon-linked sugar-containing amino acid;

a redox-active amino acid;

an α

-hydroxy containing acid;

an amino thio acid;

an α

,α

disubstituted amino acid;

a β

-amino acid;

a cyclic amino acid other than proline or histidine; and

an aromatic amino acid other than phenylalanine, tyrosine or tryptophan.

26. The cell or cell line of claim 15, wherein the tRNA recognizes a selector codon.

27. The cell or cell line of claim 26, wherein the selector codon is selected from the group consisting of:

amber codon, ochre codon, opal codon, or four or more base codons.

35. The cell or cell line of claim 15, further comprising a nucleic acid that comprises a polynucleotide that encodes a polypeptide of interest, wherein the polynucleotide comprises a selector codon that is recognized by the tRNA.

36. The cell or cell line of claim 35, wherein the polypeptide of interest is a therapeutic protein, a diagnostic protein, or an industrial enzyme.

37. The cell or cell line of claim 35, wherein the polypeptide of interest comprises a protein selected from the group consisting of:

a cytokine, a growth factor, a growth factor receptor, an interferon, an interleukin, an inflammatory molecule, an oncogene product, a peptide hormone, a signal transduction molecule, a steroid hormone receptor, erythropoietin (EPO), insulin, human growth hormone, an Alpha-1 antitrypsin, an Angiostatin, an Antihemolytic factor, an antibody, an Apolipoprotein, an Apoprotein, an Atrial natriuretic factor, an Atrial natriuretic polypeptide, an Atrial peptide, a C-X-C chemokine, T39765, NAP-2, ENA-78, a Gro-a, a Gro-b, a Gro-c, an IP-10, a GCP-2, an NAP-4, an SDF-1, a PF4, a MIG, a Calcitonin, a c-kit ligand, a CC chemokine, a Monocyte chemoattractant protein-1, a Monocyte chemoattractant protein-2, a Monocyte chemoattractant protein-3, a Monocyte inflammatory protein-1 alpha, a Monocyte inflammatory protein-1 beta, RANTES, I309, R83915, R91733, HCC1, T58847, D31065, T64262, a CD40, a CD40 ligand, a C-kit Ligand, a Collagen, a Colony stimulating factor (CSF), a Complement factor 5a, a Complement inhibitor, a Complement receptor 1, DHFR, an epithelial Neutrophil Activating Peptide-78, a GROα

/MGSA, a GROβ

, a GROγ

a MIP-1α

, a MIP-1δ

, a MCP-1, an Epidermal Growth Factor (EGF), an epithelial Neutrophil Activating Peptide, an Exfoliating toxin, a Factor IX, a Factor VII, a Factor VIII, a Factor X, a Fibroblast Growth Factor (FGF), a Fibrinogen, a Fibronectin, a G-CSF, a GM-CSF, a Glucocerebrosidase, a Gonadotropin, a Hedgehog protein, a Hemoglobin, a Hepatocyte Growth Factor (HGF), a Hirudin, a Human serum albumin, an ICAM-1, an ICAM-1 receptor, an LFA-1, an LFA-1 receptor, an Insulin-like Growth Factor (IGF), an IGF-I, an IGF-II, an IFN-α

, an IFN-β

, an IFN-γ

, an IL-1, an IL-2, an IL-3, an IL-4, an IL-5, an IL-6, an IL-7, an IL-8, an IL-9, an IL-10, an IL-11, an IL-12, a Keratinocyte Growth Factor (KGF), a Lactoferrin, a leukemia inhibitory factor, a Luciferase, a Neurturin, a Neutrophil inhibitory factor (NIF), an oncostatin M, an Osteogenic protein, a Parathyroid hormone, a PD-ECSF, a PDGF, a Pleiotropin, a Protein A, a Protein G, a Pyrogenic exotoxins A, B, or C, a Relaxin, a Renin, an SCF, a Soluble complement receptor I, a Soluble I-CAM 1, a Soluble interleukin receptor, a Soluble TNF receptor, a Somatomedin, a Somatostatin, a Somatotropin, a Streptokinase, a Superantigen, a Staphylococcal enterotoxins, an SEA, an SEB, an SEC1, an SEC2, an SEC3, an SED, an SEE, a Superoxide dismutase (SOD), a Toxic shock syndrome toxin, a Thymosin alpha 1, a Tissue plasminogen activator, a tumor growth factor (TGF), a TGF-α

, a TGF-β

, a Tumor Necrosis Factor, a Tumor Necrosis Factor alpha, a Tumor necrosis factor beta, a Tumor necrosis factor receptor (TNFR), a VLA-4 protein, a VCAM-1 protein, a Vascular Endothelial Growth Factor (VEGEF), a Urokinase, a Mos, a Ras, a Raf, a Met;

a p53, a Tat, a Fos, a Myc, a Jun, a Myb, a Rel, an estrogen receptor, a progesterone receptor, a testosterone receptor, an aldosterone receptor, an LDL receptor, a SCF/c-Kit, a CD40L/CD40, a VLA-4/VCAM-1, an ICAM-1/LFA-1, a hyalurin/CD44, and a corticosterone.

6. A tRNA transcription cassette comprising 3′

flanking sequences, nucleotides encoding human tRNA and nucleotides encoding one or more Escherichia coli or Bacillus stearothermophilus tRNA.

7. The tRNA transcription cassette from claim 6, wherein the tRNA transcription cassette also comprises a pal III promoter.

8. The tRNA transcription cassette from claim 7, wherein the pal III promoter is H1 or U6.

16. A vertebrate cell or cell line comprising the tRNA transcription cassette of claim 6.

17. The cell or cell line of claim 16, wherein the Escherichia coli or Bacillus stearothermophilus tRNA in the transcription cassette is aminoacylated with a non-natural amino acid.

22. The cell or cell line of claim 17, wherein the non-natural amino acid is selected from the group consisting of:

a p-acetyl-L-phenylalanine;

a p-iodo-L-phenylalanine;

an O-methyl-L-tyrosine;

a p-propargyloxyphenylalanine;

an L-3-(2-naphthyl)alanine;

a 3-methyl-phenylalanine;

an O-4-allyl-L-tyrosine;

a 4-propyl-L-tyrosine;

a tri-O-acetyl-GlcNAcβ

-serine;

an L-Dopa;

a fluorinated phenylalanine;

an isopropyl-L-phenylalanine;

a p-azido-L-phenylalanine;

a p-acyl-L-phenylalanine;

a p-benzoyl-L-phenylalanine;

an L-phosphoserine;

a phosphonoserine;

a phosphonotyrosine;

a p-bromophenylalanine;

a p-amino-L-phenylalanine;

an isopropyl-L-phenylalanine;

an unnatural analogue of a tyrosine amino acid;

an unnatural analogue of a glutamine amino acid;

an unnatural analogue of a phenylalanine amino acid;

an unnatural analogue of a serine amino acid;

an unnatural analogue of a threonine amino acid;

an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or any combination thereof;

an amino acid with a photoactivatable cross-linker;

a spin-labeled amino acid;

a fluorescent amino acid;

a metal binding amino acid;

a metal-containing amino acid;

a radioactive amino acid;

a photocaged and/or photoisomerizable amino acid;

a biotin or biotin-analogue containing amino acid;

a keto containing amino acid;

an amino acid comprising polyethylene glycol or polyether;

a heavy atom substituted amino acid;

a chemically cleavable or photocleavable amino acid;

an amino acid with an elongated side chain;

an amino acid containing a toxic group;

a sugar substituted amino acid;

a carbon-linked sugar-containing amino acid;

a redox-active amino acid;

an α

-hydroxy containing acid;

an amino thio acid;

an α

,α

disubstituted amino acid;

a β

-amino acid;

a cyclic amino acid other than proline or histidine; and

an aromatic amino acid other than phenylalanine, tyrosine or tryptophan.

28. The cell or cell line of claim 17, wherein the tRNA recognizes a selector codon.

29. The cell or cell line of claim 28, wherein the selector codon is selected from the group consisting of:

amber codon, ochre codon, opal codon, or four or more base codons.

38. The cell or cell line of claim 17, further comprising a nucleic acid that comprises a polynucleotide that encodes a polypeptide of interest, wherein the polynucleotide comprises a selector codon that is recognized by the tRNA.

39. The cell or cell line of claim 38, wherein the polypeptide of interest is a therapeutic protein, a diagnostic protein, or an industrial enzyme.

40. The cell or cell line of claim 38, wherein the polypeptide of interest comprises a protein selected from the group consisting of:

a cytokine, a growth factor, a growth factor receptor, an interferon, an interleukin, an inflammatory molecule, an oncogene product, a peptide hormone, a signal transduction molecule, a steroid hormone receptor, erythropoietin (EPO), insulin, human growth hormone, an Alpha-1 antitrypsin, an Angiostatin, an Antihemolytic factor, an antibody, an Apolipoprotein, an Apoprotein, an Atrial natriuretic factor, an Atrial natriuretic polypeptide, an Atrial peptide, a C-X-C chemokine, T39765, NAP-2, ENA-78, a Gro-a, a Gro-b, a Gro-c, an IP-10, a GCP-2, an NAP-4, an SDF-1, a PF4, a MIG, a Calcitonin, a c-kit ligand, a CC chemokine, a Monocyte chemoattractant protein-1, a Monocyte chemoattractant protein-2, a Monocyte chemoattractant protein-3, a Monocyte inflammatory protein-1 alpha, a Monocyte inflammatory protein-1 beta, RANTES, I309, R83915, R91733, HCC1, T58847, D31065, T64262, a CD40, a CD40 ligand, a C-kit Ligand, a Collagen, a Colony stimulating factor (CSF), a Complement factor 5a, a Complement inhibitor, a Complement receptor 1, DHFR, an epithelial Neutrophil Activating Peptide-78, a GROα

/MGSA, a GROβ

, a GROγ

a MIP-1α

, a MIP-1δ

, a MCP-1, an Epidermal Growth Factor (EGF), an epithelial Neutrophil Activating Peptide, an Exfoliating toxin, a Factor IX, a Factor VII, a Factor VIII, a Factor X, a Fibroblast Growth Factor (FGF), a Fibrinogen, a Fibronectin, a G-CSF, a GM-CSF, a Glucocerebrosidase, a Gonadotropin, a Hedgehog protein, a Hemoglobin, a Hepatocyte Growth Factor (HGF), a Hirudin, a Human serum albumin, an ICAM-1, an ICAM-1 receptor, an LFA-1, an LFA-1 receptor, an Insulin-like Growth Factor (IGF), an IGF-1, an IGF-II, an IFN-α

, an IFN-β

, an IFN-γ

, an IL-1, an IL-2, an IL-3, an IL-4, an IL-5, an IL-6, an IL-7, an IL-8, an IL-9, an IL-10, an IL-11, an IL-12, a Keratinocyte Growth Factor (KGF), a Lactoferrin, a leukemia inhibitory factor, a Luciferase, a Neurturin, a Neutrophil inhibitory factor (NIF), an oncostatin M, an Osteogenic protein, a Parathyroid hormone, a PD-ECSF, a PDGF, a Pleiotropin, a Protein A, a Protein G, a Pyrogenic exotoxins A, B, or C, a Relaxin, a Renin, an SCF, a Soluble complement receptor I, a Soluble I-CAM 1, a Soluble interleukin receptor, a Soluble TNF receptor, a Somatomedin, a Somatostatin, a Somatotropin, a Streptokinase, a Superantigen, a Staphylococcal enterotoxins, an SEA, an SEB, an SEC1, an SEC2, an SEC3, an SED, an SEE, a Superoxide dismutase (SOD), a Toxic shock syndrome toxin, a Thymosin alpha 1, a Tissue plasminogen activator, a tumor growth factor (TGF), a TGF-α

, a TGF-β

, a Tumor Necrosis Factor, a Tumor Necrosis Factor alpha, a Tumor necrosis factor beta, a Tumor necrosis factor receptor (TNFR), a VLA-4 protein, a VCAM-1 protein, a Vascular Endothelial Growth Factor (VEGEF), a Urokinase, a Mos, a Ras, a Raf, a Met;

a p53, a Tat, a Fos, a Myc, a Jun, a Myb, a Rel, an estrogen receptor, a progesterone receptor, a testosterone receptor, an aldosterone receptor, an LDL receptor, a SCF/c-Kit, a CD40L/CD40, a VLA-4/VCAM-1, an ICAM-1/LFA-1, a hyalurin/CD44, and a corticosterone.

9. A tRNA transcription cassette comprising 5′

flanking sequences, 3′

flanking sequences, nucleotides encoding human tRNA and nucleotides encoding one or more Escherichia coli or Bacillus stearothermophilus tRNA.

10. The tRNA transcription cassette from claim 9, wherein the tRNA transcription cassette also comprises a pol III promoter.

11. The tRNA transcription cassette from claim 10, wherein the pol III promoter is H1 or U6.

18. A vertebrate cell or cell line comprising the tRNA transcription cassette of claim 9.

19. The cell or cell line of claim 18, wherein the Escherichia coli or Bacillus stearothermophilus tRNA in the transcription cassette is aminoacylated with a non-natural amino acid.

23. The cell or cell line of claim 19, wherein the non-natural amino acid is selected from the group consisting of:

a p-acetyl-L-phenylalanine;

a p-iodo-L-phenylalanine;

an O-methyl-L-tyrosine;

a p-propargyloxyphenylalanine;

an L-3-(2-naphthyl)alanine;

a 3-methyl-phenylalanine;

an O-4-allyl-L-tyrosine;

a 4-propyl-L-tyrosine;

a tri-O-acetyl-GlcNAcβ

-serine;

an L-Dopa;

a fluorinated phenylalanine;

an isopropyl-L-phenylalanine;

a p-azido-L-phenylalanine;

a p-acyl-L-phenylalanine;

a p-benzoyl-L-phenylalanine;

an L-phosphoserine;

a phosphonoserine;

a phosphonotyrosine;

a p-bromophenylalanine;

a p-amino-L-phenylalanine;

an isopropyl-L-phenylalanine;

an unnatural analogue of a tyrosine amino acid;

an unnatural analogue of a glutamine amino acid;

an unnatural analogue of a phenylalanine amino acid;

an unnatural analogue of a serine amino acid;

an unnatural analogue of a threonine amino acid;

an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or any combination thereof;

an amino acid with a photoactivatable cross-linker;

a spin-labeled amino acid;

a fluorescent amino acid;

a metal binding amino acid;

a metal-containing amino acid;

a radioactive amino acid;

a photocaged and/or photoisomerizable amino acid;

a biotin or biotin-analogue containing amino acid;

a keto containing amino acid;

an amino acid comprising polyethylene glycol or polyether;

a heavy atom substituted amino acid;

a chemically cleavable or photocleavable amino acid;

an amino acid with an elongated side chain;

an amino acid containing a toxic group;

a sugar substituted amino acid;

a carbon-linked sugar-containing amino acid;

a redox-active amino acid;

an α

-hydroxy containing acid;

an amino thio acid;

an α

,α

disubstituted amino acid;

a β

-amino acid;

a cyclic amino acid other than proline or histidine; and

an aromatic amino acid other than phenylalanine, tyrosine or tryptophan.

30. The cell or cell line of claim 19, wherein the tRNA recognizes a selector codon.

31. The cell or cell line of claim 30, wherein the selector codon is selected from the group consisting of:

amber codon, ochre codon, opal codon, or four or more base codons.

41. The cell or cell line of claim 19, further comprising a nucleic acid that comprises a polynucleotide that encodes a polypeptide of interest, wherein the polynucleotide comprises a selector codon that is recognized by the tRNA.

42. The cell or cell line of claim 41, wherein the polypeptide of interest is a therapeutic protein, a diagnostic protein, or an industrial enzyme.

43. The cell or cell line of claim 41, wherein the polypeptide of interest comprises a protein selected from the group consisting of:

a cytokine, a growth factor, a growth factor receptor, an interferon, an interleukin, an inflammatory molecule, an oncogene product, a peptide hormone, a signal transduction molecule, a steroid hormone receptor, erythropoietin (EPO), insulin, human growth hormone, an Alpha-1 antitrypsin, an Angiostatin, an Antihemolytic factor, an antibody, an Apolipoprotein, an Apoprotein, an Atrial natriuretic factor, an Atrial natriuretic polypeptide, an Atrial peptide, a C-X-C chemokine, T39765, NAP-2, ENA-78, a Gro-a, a Gro-b, a Gro-c, an IP-10, a GCP-2, an NAP-4, an SDF-1, a PF4, a MIG, a Calcitonin, a c-kit ligand, a CC chemokine, a Monocyte chemoattractant protein-1, a Monocyte chemoattractant protein-2, a Monocyte chemoattractant protein-3, a Monocyte inflammatory protein-1 alpha, a Monocyte inflammatory protein-1 beta, RANTES, I309, R83915, R91733, HCC1, T58847, D31065, T64262, a CD40, a CD40 ligand, a C-kit Ligand, a Collagen, a Colony stimulating factor (CSF), a Complement factor 5a, a Complement inhibitor, a Complement receptor 1, DHFR, an epithelial Neutrophil Activating Peptide-78, a GROα

/MGSA, a GROβ

, a GROγ

a MIP-1α

, a MIP-18, a MCP-1, an Epidermal Growth Factor (EGF), an epithelial Neutrophil Activating Peptide, an Exfoliating toxin, a Factor IX, a Factor VII, a Factor VIII, a Factor X, a Fibroblast Growth Factor (FGF), a Fibrinogen, a Fibronectin, a G-CSF, a GM-CSF, a Glucocerebrosidase, a Gonadotropin, a Hedgehog protein, a Hemoglobin, a Hepatocyte Growth Factor (HGF), a Hirudin, a Human serum albumin, an ICAM-1, an ICAM-1 receptor, an LFA-1, an LFA-1 receptor, an Insulin-like Growth Factor (IGF), an IGF-I, an IGF-II, an IFN-α

, an IFN-β

, an IFN-γ

, an IL-1, an IL-2, an IL-3, an IL-4, an IL-5, an IL-6, an IL-7, an IL-8, an IL-9, an IL-10, an IL-11, an IL-12, a Keratinocyte Growth Factor (KGF), a Lactoferrin, a leukemia inhibitory factor, a Luciferase, a Neurturin, a Neutrophil inhibitory factor (NIF), an oncostatin M, an Osteogenic protein, a Parathyroid hormone, a PD-ECSF, a PDGF, a Pleiotropin, a Protein A, a Protein G, a Pyrogenic exotoxins A, B, or C, a Relaxin, a Renin, an SCF, a Soluble complement receptor I, a Soluble I-CAM 1, a Soluble interleukin receptor, a Soluble TNF receptor, a Somatomedin, a Somatostatin, a Somatotropin, a Streptokinase, a Superantigen, a Staphylococcal enterotoxins, an SEA, an SEB, an SEC1, an SEC2, an SEC3, an SED, an SEE, a Superoxide dismutase (SOD), a Toxic shock syndrome toxin, a Thymosin alpha 1, a Tissue plasminogen activator, a tumor growth factor (TGF), a TGF-α

c, a TGF-β

, a Tumor Necrosis Factor, a Tumor Necrosis Factor alpha, a Tumor necrosis factor beta, a Tumor necrosis factor receptor (TNFR), a VLA-4 protein, a VCAM-1 protein, a Vascular Endothelial Growth Factor (VEGEF), a Urokinase, a Mos, a Ras, a Raf, a Met;

a p53, a Tat, a Fos, a Myc, a Jun, a Myb, a Rel, an estrogen receptor, a progesterone receptor, a testosterone receptor, an aldosterone receptor, an LDL receptor, a SCF/c-Kit, a CD40L/CD40, a VLA-4/VCAM-1, an ICAM-1/LFA-1, a hyalurin/CD44, and a corticosterone.