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, the host cell comprising a vector having a polynucleotide encoding the target polypeptide,    (b) site-specifically incorporating one or more non-natural amino acid codons into the polynucleotide, wherein at least one non-natural amino acid codon corresponds to the first position of the amino terminus of the target polypeptide,    (c) growing the host cell under conditions such that the host cell expresses the target polypeptide, wherein the target molecule retains the non-natural amino acid residue at the first position of the amino terminus, and wherein the non-natural amino acid residue at the first position of the amino terminus contains an azide, alkyne, vinyl, or aryl halide group, thereby producing a modified target polypeptide.    
     
     
         2 . The method of  claim 1  wherein one or more non-natural amino acid codon encodes the penultimate position of the amino terminus of the target polypeptide.  
     
     
         3 . The method of claims  1  or  2  wherein the one or more non-natural amino acids 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-idiophenylalanine, p-azidophenylalanine, 3-(6-chloroindolyl)alanine, 3-(6-bromoindolyl)alanine, 3-(5-bromoindolyl)alanine, azidohomoalanine, and p-chlorophenylalanine.  
     
     
         4 . The method of  claim 1  further comprising attaching a chemical moiety to one or more of the non-natural amino acid residues in the target polypeptide.  
     
     
         5 . The method of  claim 4  wherein the chemical moiety is attached to the non-natural amino acid residue in the first position of the amino terminus of the target polypeptide.  
     
     
         6 . The method of  claim 5  wherein the non-natural amino acid is fluorinated, electroactive, or unsaturated.  
     
     
         7 . The method of  claim 5  wherein the chemical moiety is attached to the non-natural amino acid residue by a single carbon-carbon linkage, a double carbon-carbon linkage, a triple carbon-carbon linkage, or a triazole linkage between the non-natural amino acid and the chemical moiety.  
     
     
         8 . The method of  claim 5  wherein the chemical moiety is attached to the non-natural amino acid residue by a covalent interaction.  
     
     
         9 . The method of  claim 5  wherein the chemical moiety is attached to the non-natural amino acid residue by way of a chemical reaction selected from the group consisting of: copper catalyzed [3+2]cycloaddition, Suzuki coupling, Hiyama coupling, Kumada coupling, Heck reaction, Cadiot-Chodkiewicz coupling, and Sonogashira coupling.  
     
     
         10 . The method of  claim 5  wherein the chemical moiety is selected from the group consisting of: cytotoxins, pharmaceutical drugs, dyes or fluorescent labels, a nucleophilic or electrophilic group, a ketone or aldehyde, azide or alkyne compounds, photocaged groups, tags, a peptide, a polypeptide, a protein, an oligosaccharide, polyethylene glycol with any molecular weight and in any geometry, polyvinyl alcohol, metals, metal complexes, polyamines, imidizoles, carbohydrates, lipids, biopolymers, particles, solid supports, a polymer, a targeting agent, an affinity group, any agent to which a complementary reactive chemical group can be attached, biophysical or biochemical probes, isotypically-labeled probes, spin-label amino acids, fluorophores, aryl iodides and bromides.  
     
     
         11 . The method of  claim 1  wherein the target polypeptide is selected from the group consisting of: an antibody, antibody fragment, antibody derivative, Fab, Fab′, F(ab)2, Fd, Fv, ScFv, diabody, tribody, tetrabody, dimer, trimer or minibody, a cytokine, a transcriptional modulator that modulates cell growth, differentiation or regulation, expression activator, inflammatory molecule, growth factor, growth factor receptor, and oncogene product.  
     
     
         12 . The method of  claim 11 , wherein the target polypeptide is selected from the group consisting of: Factor VII, Factor VII, Factor IX, Follitropin, thrombopoeitin, erythropoietin, human growth hormone, G-CSF, GM-CSF, interferon-α, interferon-β, interferon-γ, interferon-Ω, interferon-τ, and GLP-1.  
     
     
         13 . The method of  claim 1  wherein the site-specifically incorporating one or more amino acid codons is conducted by a technique 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, use of an external mutant aminoacyl-tRNA synthetase, and incorporation of a bias codon.  
     
     
         14 . A composition comprising a modified target polynucleotide encoding a target polypeptide, the target polynucleotide comprising one or more non-natural amino acid codons wherein at least one non-natural amino acid codon contains an azide, alkyne, vinyl, or aryl halide group and corresponds to the first position of the amino terminus of the target polypeptide.  
     
     
         15 . The composition of  claim 14  further comprising a host cell.  
     
     
         16 . The composition of  claim 14  wherein at least one non-natural amino acid codon corresponds to the penultimate position of the amino terminus of the target polypeptide.  
     
     
         17 . The composition of  claim 14  further comprising a chemical moiety attached to one or more non-natural amino acid residues in the target polypeptide.  
     
     
         18 . The composition of  claim 14  wherein the chemical moiety is attached at least to the non-natural amino acid residue in the first position of the amino terminus of the target polypeptide.  
     
     
         19 . The composition of  claim 18  wherein the chemical moiety is covalently attached to the non-natural amino acid corresponding to the first position of the amino terminus of the target polypeptide.  
     
     
         20 . The composition of  claim 18  wherein the chemical moiety is attached to the non-natural amino acid corresponding to the first position of the amino terminus of the target polypeptide by a single carbon-carbon linkage, a double carbon-carbon linkage, a triple carbon-carbon linkage, or a triazole linkage between the chemical moiety and the non-natural amino acid.  
     
     
         21 . The composition of  claim 19  wherein the chemical moiety is selected from the group consisting of: cytotoxins, pharmaceutical drugs, dyes or fluorescent labels, a nucleophilic or electrophilic group, a ketone or aldehyde, azide or alkyne compounds, photocaged groups, tags, a peptide, a polypeptide, a protein, an oligosaccharide, polyethylene glycol with any molecular weight and in any geometry, polyvinyl alcohol, metals, metal complexes, polyamines, imidizoles, carbohydrates, lipids, biopolymers, particles, solid supports, a polymer, a targeting agent, an affinity group, any agent to which a complementary reactive chemical group can be attached, biophysical or biochemical probes, isotypically-labeled probes, spin-label amino acids, fluorophores, aryl iodides and bromides.  
     
     
         22 . The composition of  claim 20  wherein the modified target polypeptide is selected from the group consisting of: an antibody, antibody fragment, antibody derivative, Fab, Fab′, F(ab)2, Fd, Fv, ScFv, diabody, tribody, tetrabody, dimer, trimer or minibody, a cytokine, a transcriptional modulator that modulates cell growth, differentiation, or regulation, expression activator, inflammatory molecule, growth factor, growth factor receptor, and oncogene product.  
     
     
         23 . The composition of  claim 21 , wherein the modified target polypeptide is selected from the group consisting of: Factor VII, Factor VIII, Factor IX, Follitropin, thrombopoeitin, erythropoietin, human growth hormone, G-CSF, GM-CSF, interferon-α, interferon-β, interferon-γ, interferon-Ω, interferon-τ, and GLP-1.  
     
     
         24 . The composition of  claim 22  wherein the modified target polypeptide comprises interferon-β.  
     
     
         25 . The composition of  claim 23  wherein at least one of the non-natural amino acid codons corresponds to positions selected from the group consisting of: 2, 17, 36, 40, 44, 62, and 117 of the modified target polypeptide.  
     
     
         26 . A pharmaceutical composition comprising a modified target polypeptide comprising a target polypeptide having one or more non-natural amino acids residues incorporated, wherein at least one of the non-natural amino acid residues corresponds to the first position of the amino terminus of the target polypeptide.  
     
     
         27 . A method for preparing a refolded, soluble form of an insoluble or aggregated PEGylated interferon beta protein comprising one or more free cysteine residues, said method comprising the steps of: 
 (a) causing a host cell to express a interferon beta protein in an insoluble or aggregated form;    (b) lysing the cells by chemical, enzymatic or physical means;    (c) solubilizing the insoluble or aggregated protein by exposing the insoluble or aggregated protein to a denaturing agent, a reducing agent and a cysteine blocking agent;    (d) PEGylating the reduced, denatured protein, and    (e) refolding the PEGylated protein by reducing the concentrations of the denaturing agent and reducing agents to levels sufficient to allow the PEG interferon beta protein to refold into a soluble, biologically active form.