In vivo incorporation of unnatural amino acids

Abstract

The invention provides methods and compositions for in vivo incorporation of unnatural amino acids. Also provided are compositions including proteins with unnatural amino acids.

1 - 47 . (canceled)  
     
     
         48 . A method for producing, in a translation system, at least a first protein comprising at least one unnatural amino acid, the method comprising: 
 providing the translation system with at least one nucleic acid comprising at least one selector codon, wherein the nucleic acid encodes the first protein;    providing the translation system with an orthogonal tRNA (O-tRNA), wherein the O-tRNA recognizes the at least one selector codon;    providing the translation system with an orthogonal aminoacyl tRNA synthetase (O-RS), wherein the O-RS preferentially aminoacylates the O-tRNA with the at least one unnatural amino acid in the translation system; and,    translating the nucleic acid in the translation system in the presence of the unnatural amino acid, the O-tRNA and the O-RS, whereby the unnatural amino acid is incorporated into the at least one protein.    
     
     
         49 . The method of  claim 48 , wherein the translation system comprises a cell.  
     
     
         50 . The method of  claim 49 , wherein the translation system comprises a bacterial cell.  
     
     
         51 . The method of  claim 50 , wherein the translation system comprises an  Escherichia coli  cell.  
     
     
         52 . The method of  claim 49 , wherein the translation system comprises an archeaebacterial cell.  
     
     
         53 . The method of  claim 49 , wherein the translation system comprises a eukaryotic cell.  
     
     
         54 . The method of  claim 53 , wherein the eukaryotic cell comprises a yeast cell, a mammalian cell, a plant cell, or an insect cell.  
     
     
         55 . (canceled)  
     
     
         56 . The method of  claim 49 , wherein the first protein is homologous to a second protein selected from the group consisting of: an angiostatin, an antihemolytic factor, an antibody, an apolipoprotein, an apoprotein, an atrial natriuretic factor, an atrial natriuretic polypeptide, an atrial peptide, a CC chemokine, a C—X—C chemokine, a cytokine, an epithelial neutrophil activating peptide, 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, a transcriptional activator, a transcriptional suppressor, a soluble interleukin receptor.  
     
     
         57 . The method of  claim 49 , wherein the first protein is homologous to a second protein selected from the group consisting of: 
 an aldosterone receptor, an alpha-1 antitrypsin, a corticosterone, a calcitonin, a CD40, a CD40 ligand, CD44, a c-kit ligand, a collagen, a colony stimulating factor (CSF), a complement factor 5a, a complement inhibitor, a complement receptor 1, a corticosterone, D31065, an ENA-78, an epidermal growth factor (EGF), an epithelial neutrophil activating peptide-78, an erythropoietin (EPO), an estrogen receptor, an exfoliating toxin, Factor VII, a Factor VIII, a Factor IX, a Factor X, a fibroblast growth factor (FGF), a fibrinogen, a Fibronectin, a fos, a GCP-2, a G-CSF, a Glucocerebrosidase, a GM-CSF, a Gonadotropin, a GROα/MGS, a GROα/MGSA, a Gro-a, a Gro-b, a Gro-c, a GROβ, a GROγ, a GROγ, an HCC1, a hedgehog protein, a hemoglobin, an hepatocyte growth factor (HGF), an hirudin, a human growth hormone, a human serum albumin, a hyalurin, an I309, an ICAM-1, an ICAM-1 receptor, an ICAM-1/LFA-1, an IFN-α, an IFN-β, an IFN-γ, an IGF-I, and IGF-II, 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, an insulin, an insulin-like growth factor (IGF), an IP-10, a Jun, a Keratinocyte Growth Factor (KGF), a Lactoferrin, an LDL receptor, a leukemia inhibitory factor, an LFA-1, an LFA-1 receptor, a Luciferase, an MCP-1, a Met, a MIG, an MIP-1β, a MIP-1α, a MIP-1 , a Monocyte chemoattractant protein-1, a Monocyte chemoattractant protein-2, a Monocyte chemoattractant protein-3, Monocyte inflammatory protein-1 alpha, a Monocyte inflammatory protein-1 beta, a Mos, a Myb, a Myc, a NAP-2, a NAP-4, a Neurturin, a Neutrophil inhibitory factor (NIF), an oncostatin M, an osteogenic protein, a p53, a parathyroid hormone, a PD-ECSF, a PDGF, a PF4, a pleiotropin, a progesterone receptor, a protein A, a protein G, a pyrogenic exotoxin A, a pyrogenic exotoxin B, a pyrogenic exotoxin C, an R83915, an R91733, a Raf, a RANTES, a Ras, a Rel, a Relaxin, a Renin, an SCF, an SDF-1, an SEA, an SEB, an SEC1, an SEC2, an SEC3, a SED, an SEE, a soluble complement receptor I, a Soluble I-CAM 1, a soluble TNF receptor, a somatomedin, a somatostatin, a somatotropin, a staphylococcal enterotoxin, a steroid hormone receptor, a streptokinase, a superantigen, a superoxide dismutase, a T58847, a T64262, a Tat, a testosterone receptor, a TGF-α, a TGF-β, a thymosin alpha 1, a tissue plasmino en activator, a tumor Necrosis Factor (TNF), a toxic shock syndrome toxin, a tumor growth factor (TGF), a TNF-alpha, a TNF-beta, a tumor necrosis factor receptor (TNFR), a urokinase, a vascular endothelial growth factor (VEGEF), a VCAM-1 protein, and a VLA-4 protein.    
     
     
         58 . The method of  claim 57 , wherein the first protein is at least about 50% identical to the second protein.  
     
     
         59 . The method of  claim 57 , wherein the second protein is a naturally occuring erythropoeitin (EPO) and the protein at least about 50% identical to a naturally occuring EPO.  
     
     
         60 . The protein of  claim 55 , wherein the first protein comprises at least two unnatural amino acids.  
     
     
         61 . The protein of  claim 55 , wherein the first protein comprises at least three unnatural amino acids.  
     
     
         62 . The protein of  claim 55 , wherein the first protein comprises at least four unnatural amino acids.  
     
     
         63 . The protein of  claim 55 , wherein the first protein comprises at least five or more unnatural amino acids.  
     
     
         64 . The method of  claim 48 , wherein the translation system comprises an in vitro translation system.  
     
     
         65 . The method of  claim 64 , wherein the translation system comprises a cell extract.  
     
     
         66 . The method of  claim 48  wherein the first protein is a Asp112TAG mutant of chloramphenicol acetylransferase (CAT), a Tyr163TAG mutant of mouse dihydrofolate reductase (DHFR), or a Tyr163TAG mutant of mouse dihydrofolate reductase (DHFR) comprising a COOH His6tag.  
     
     
         67 . The method of  claim 48  wherein the unnatural amino acid is selected from the group consisting of: 
 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-iodo-phenylalanine, 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; an amino acid with a novel functional group; an amino acid that covalently or noncovalently interacts with another molecule; 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 glycosylated or carbohydrate modified amino acid; a keto containing amino acid; amino acids 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, e.g., a sugar substituted serine or the like; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an α-hydroxy containing acid; an amino thio acid containing amino acid; an α,α disubstituted amino acid; a β-amino acid; and a cyclic amino acid other than proline.    
     
     
         68 . The method of  claim 48  wherein the at least one unnatural amino acid is a O-methyl-L-tyrosine.  
     
     
         69 . The method of  claim 48  wherein the at least one unnatural amino acid is an L-3-(2-naphthyl)alanine.  
     
     
         70 . The method of  claim 48  wherein the at least one unnatural amino acid is an amino-, isopropyl-, or O-allyl-containing phenylalanine analogue.  
     
     
         71 . The method of  claim 48 , wherein the O-tRNA is transcribed from a nucleic acid comprising a polynucleotide sequence selected from the group consisting of: SEQ ID NO:1-3, and a complementary polynucleotide sequence thereof.  
     
     
         72 . The method of  claim 48 , wherein the at least one selector codon is stop codon, a rare codon, or a four base codon.  
     
     
         73 . The method of  claim 72 , wherein the stop codon is an amber codon.  
     
     
         74 . The method of  claim 48 , wherein the O-RS preferentially aminoacylates the O-tRNA with an O-methyl-L-tyrosine.  
     
     
         75 . The method of  claim 48 , wherein the O-RS preferentially aminoacylates the O-tRNA with an amino-, isopropyl-, or O-allyl-containing phenylalanine analogue.  
     
     
         76 . The method of  claim 48 , wherein the O-RS comprises a polypeptide selected from the group consisting of: 
 a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 36-66.    
     
     
         77 . The method of  claim 48  wherein the unnatural amino acid is provided exogenously.  
     
     
         78 . The method of  claim 48  wherein the translation system is a cell and the unnatural amino acid is biosynthesized by the cell.  
     
     
         79 - 140 . (canceled)