Amino acid substituted molecules

Abstract

The invention provides compositions and methods of identifying, modifying and producing modified target molecules, including therapeutic molecules by modification with non-natural amino acids. Certain aspects of the invention include methods of adding a chemical moiety to a target molecule, and the compositions resulting therefrom. Certain aspects of the invention also relate to kits for identifying, modifying and producing modified target molecules described herein.

1 . A method for producing a modified target polypeptide, comprising:
 (a) providing a host cell comprising a vector, wherein said vector comprises a polynucleotide encoding a target polypeptide;   (b) site-specifically incorporating one or more non-natural amino acid residue(s) into the target polypeptide to produce a modified target polypeptide, wherein:
 (i) one or more codons encoding naturally occurring amino acids are incorporated into said polynucleotide; 
 (ii) the one or more non-natural amino acid(s) are incorporated into the target polypeptide at one or more natural amino acid position(s) encoded by the one or more codon(s) of (i) or one or more unchanged codon(s) encoding a natural amino acid; and 
   (c) growing the host cell under conditions such that the host cell expresses the modified target polypeptide.   
     
     
         2 . The method of  claim 1 , wherein the modified target polypeptide comprises one or more non-natural amino acid(s) site-specifically incorporated into the target polypeptide at one or more natural amino acid position(s) corresponding to (i). 
     
     
         3 . The method of  claim 1 , wherein said host cell is an auxotroph. 
     
     
         4 . The method of  claim 1 , wherein said site-specific incorporation of (b) occurs by protein translation in the host cell. 
     
     
         5 . The method of  claim 4 , wherein said protein translation occurs either by mutant transcription machinery or mutant translation machinery, or both. 
     
     
         6 . The method of  claim 5 , wherein said mutant transcription machinery comprises one or more mutant tRNA(s) or mutant amino-acyl tRNA synthetase(s). 
     
     
         7 . The method of  claim 1 , wherein the one or more non-natural amino acid residues(s) of (ii) replace(s) one or more methionine, tryptophan, isoleucine or leucine residue. 
     
     
         8 . The method of  claim 1 , wherein the target polypeptide is an interferon beta. 
     
     
         9 . The method of  claim 1 , wherein the target polypeptide is selected from the group consisting of: α-1 antitrypsin, Angiostatin, Antihemolytic factor, an antibody, an antibody fragment, an antibody derivative, a Fab, aFab′, aF(ab) 2 , a Fd, a Fv, a ScFv, a diabody, a tribody, a tetrabody, a dimer, a trimer, a minibody, an angiogenic molecule, an angiostatic molecule, Apolipoprotein, Apoprotein, Atrial natriuretic factor, Atrial natriuretic polypeptide, an Atrial peptide, a C-X-C chemokine, Calcitonin, a CC chemokine, CD40 ligand, C-kit ligand, Chloramphenical acetyltransferase, a Collagen, a Colony stimulating factor, Complement factor 5a, Complement inhibitor, Complement receptor 1, a cytokine, an Epidermal Growth Factor, Erythropoietin, Exfoliating toxins A and B, Factor IX, Factor VII, Factor VIII, Factor X, a Fibroblast Growth Factor, Fibrinogen, Fibronectin, Follitropin, G-CSF, GM-CSF, Glucocerebrosidase, Gonadotropin, growth factor, Hedgehog protein, Hemoglobin, Hepatocyte Growth Factor, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hirudin, Human serum albumin, Human Growth Hormone, Insulin, Insulin-like Growth Factor, GM-CSF, G-CSF, M-CSF, IFN-α, IFN-β, IFN-γ, IFN-Ω, IFN-τ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, Keratinocyte Growth Factor, Lactoferrin, leukemia inhibitory factor, Luciferase, Neurturin, Neutrophil inhibitory factor, oncostatin M, Osteogenic protein, Parathyroid hormone, PD-ECSF, PDGF, peptide hormones, Phenylalanine hydroxylase, Urikase, Pleiotropin, Protein A, Protein G, Pyrogenic exotoxins A, B, and C, Relaxin, Renin, SCF, Soluble complement receptor I, Soluble I-CAM 1, Soluble interleukin receptors, Soluble TNF receptor, soluble adhesion molecules, Somatomedin, Somatostatin, Somatotropin, Streptokinase, Superantigen, Staphylococcal enterotoxin, Superoxide dismutase, Toll-like receptors, Toxic shock syndrome toxin, Thymosin α1, Tissue plasminogen activator, Tumor necrosis factor β, Tumor necrosis factor receptor, Tumor necrosis factor-α, Vascular Endothelial Growth Factor, a virus-like particle, Urokinase; a transcriptional modulator that modulates cell growth, differentiation, or regulation, an expression activator, an inflammatory molecule, a growth factor, a growth factor receptor, an oncogene product, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α, TGF-β, EGF, KGF, SCF/c-Kit, CD40L/CD40, VLA-4/VCAM-1, ICAM-1/LFA-1, hyalurin/CD44, a signal transduction molecule, a transcriptional activator, a transcriptional suppressor, estrogen, progesterone, testosterone, aldosterone, LDL, corticosterone amidase, amino acid racemase, an acylase, a dehalogenase, a dioxygenase, a diarylpropane peroxidase, an epimerase, epoxide hydrolase, an esterase, an isomerase, a kinase, a glucose isomerase, a glycosidase, a glycosyl transferase, a haloperoxidase, a monooxygenase, a lipase, a lignin peroxidase, nitrile hydratase, nitrilase, protease, a phosphatase, subtilisin, a transaminase, and a nuclease. 
     
     
         10 . The method of  claim 1 , wherein the non-natural amino acid residue(s) comprise(s) a side chain having one or more functional group(s) not present in a natural amino acid side chain. 
     
     
         11 . The method of  claim 10 , wherein the one or more functional group(s) is selected from the group consisting of: bromo-, iodo-, ethynyl-, cyano-, azido-acetyl, aryl ketone, ketone, aldeyhyde, alkenyl-, an azide group, an alkyne group, a vinyl group, an aryl halide group, a photolabile group, a fluorescent group, and a heavy metal. 
     
     
         12 . The method of  claim 1 , wherein the one or more non-natural amino acid residue(s) is selected from the group consisting of: azidonorleucine, 3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine, p-ethynyl-phenylalanine, p-propargly-oxy-phenylalanine, m-ethynyl-phenylalanine, 6-ethynyl-tryptophan, 5-ethynyl-tryptophan, (R)-2-amino-3-(4-ethynyl-1H-pyrol-3-yl)propanic acid, p-bromophenylalanine, p-iodophenylalanine, p-azidophenylalanine, p-acetylphenylalanine, 3-(6-chloroindolyl)alanine, 3-(6-bromoindolyl)alanine, 3-(5-bromoindolyl)alanine, azidohomoalanine, homopropargylglycine, p-chlorophenylalanine, α-aminocaprylic acid, O-methyl-L-tyrosine, N-acetylgalactosamine-α-threonine, and N-acetylgalactosamine-α-serine. 
     
     
         13 . The method of  claim 1 , wherein said site-specific incorporation of (b) occurs by a method selected from the group consisting of: site-directed mutagenesis, error-prone PCR, gene shuffling, homologous recombination, incorporation of an amber stop codon, incorporation of a wobble codon, and incorporation of a bias codon. 
     
     
         14 . The method of  claim 1 , further comprising attaching one or more chemical moieties to one or more of said one or more non-natural amino acid residue(s). 
     
     
         15 . The method of  claim 14 , wherein the one or more chemical moieties is selected from the group consisting of: cytotoxins, pharmaceutical drugs, dyes, fluorescent labels, a nucleophilic group, an electrophilic group, a ketone, an aldehyde, an azide, an alkyne, photocaged groups, tags, a peptide, a polypeptide, a protein, an oligosaccharide, a polyethylene glycol of any molecular weight and in any geometry, a polyvinyl alcohol, a polyaklylene oxide, metals, metal complexes, polyamines, imidazoles, carbohydrates, lipids, biopolymers, particles, solid supports, a polymer, a water-soluble polymer, a targeting agent, an affinity group, any agent to which a complementary reactive chemical group can be attached, biophysical probes, biochemical probes, isotypically-labeled probes, spin-label amino acids, fluorophores, aryl iodides and bromides. 
     
     
         16 . A composition comprising a modified molecule comprising one or more amino acid residues substituted with a different naturally occurring amino acid residue, one or more non-natural amino acid residues, and an excipient, wherein said modified molecule has sustained in vivo retention.