OVEREXPRESSION OF AMINOACYL-tRNA SYNTHETASES FOR EFFICIENT PRODUCTION OF ENGINEERED PROTEINS CONTAINING AMINO ACID ANALOGUES

Abstract

Methods for producing modified polypeptides containing amino acid analogues are disclosed. The invention further provides purified dihydrofolate reductase polypeptides, produced by the methods of the invention, in which the methionine residues have been replaced with homoallylglycine, homoproparglycine, norvaline, norleucine, cis-crotylglycine, trans-crotylglycine, 2-aminoheptanoic acid, 2-butynylglycine and allylglycine.

What is claimed: 
     
         1 . A method for producing a modified polypeptide, comprising:
 a. providing a host cell in a medium, the host cell comprising:
 i. a vector having a polynucleotide sequence encoding an aminoacyl-tRNA synthetase for an amino acid analogue; and 
 ii. a vector having a polynucleotide sequence encoding a polypeptide molecule of interest so as to produce a host vector system; wherein the vectors of (i) and (ii) may be the same or different, 
   b. replacing the medium with a medium which has the desired amino acid analogue or adding the desired amino acid analogue to the medium, wherein the desired amino acid analogue is selected from the group consisting of an analogue that comprises side chain functionalities different from its corresponding natural amino acid, an analogue that is an optical isomer of the corresponding natural amino acid, an analogue that is a hydrophobic amino acid analogue, and an analogue that comprises fluorinated, electroactive, conjugated, azido, carbonyl, alkyl, or unsaturated side chain functionalities; and   c. growing the host cell in the medium which has the desired amino acid analogue under conditions so that the host cell expresses the polypeptide molecule of interest and the desired amino acid analogue is incorporated into the polypeptide molecule of interest thereby producing the modified polypeptide.   
     
     
         2 . The method of  claim 1 , wherein the vector having a polynucleotide sequence encoding an aminoacyl-tRNA synthetase and the vector having a polynucleotide sequence encoding a polypeptide of interest are the same vector. 
     
     
         3 . The method of  claim 1 , wherein the vector having a polynucleotide sequence encoding an aminoacyl-tRNA synthetase and the vector having a polynucleotide sequence encoding a polypeptide of interest may independently comprise an inducible or constitutive promoter. 
     
     
         4 . The method of  claim 1 , wherein the host cell is an auxotrophic host cell. 
     
     
         5 . The method of  claim 4 , wherein the auxotrophic host cell is from an organism that is selected from the group consisting of: bacteria, yeast, mammal, insect, and plant. 
     
     
         6 . The method of  claim 1 , wherein the polynucleotide sequence encoding an aminoacyl-tRNA synthetase originated from a different cell than the host cell. 
     
     
         7 . A recombinant vector comprising a polynucleotide sequence encoding an aminoacyl-tRNA synthetase for an amino acid analogue and a polynucleotide sequence encoding a polypeptide molecule of interest. 
     
     
         8 . The vector of  claim 7 , further comprising at least one expression element. 
     
     
         9 . The vector of  claim 8 , wherein at least one expression element is selected from the group consisting of: promoter sequence, secretion signal, enhancer sequence, transcription terminator, Shine-Dalgarno sequence, initiator codon, and termination codon. 
     
     
         10 . The vector of  claim 7 , wherein the polynucleotide sequence encodes an aminoacyl-tRNA synthetase that originated from a different cell than the host cell. 
     
     
         11 . A composition comprising the vector of  claim 7 , and a host cell. 
     
     
         12 . The composition of  claim 11 , wherein the host cell is an auxotrophic host cell. 
     
     
         13 . The composition of  claim 12 , wherein the auxotrophic host cell is from an organism that is selected from the group consisting of: bacteria, yeast, mammal, insect, and plant. 
     
     
         14 . The method of  claim 1 , wherein the amino acid analogue is selected from the group consisting of 6,6,6-trifluoromethionine, homoallyglycine, homoproparglycine, norvaline, norleucine, cis-crotylglycine, trans-crotylglycine, 2-aminoheptanoic acid, 2-butynylglycine, allylglycine, azidoalanine, 2-aminoethylcysteine, o-methylthreonine, gamma-methylleucine, beta-methylvaline, alloisoleucine, beta-fluoroasparagine, pyridylalanine, p-aminophenylalanine, dehydroalanine, beta-methylenenorvaline, N-methylalanine, alpha-difluoromethyllysine, p-methoxy-m-hydroxyphenylalanine, furanomycin, azidohomoalanine, o-allylserine, and propargylglycine, selenomethionine, telluromethionine, ethionine, naphthylalanine, and amino acids with sides chains containing divalent non-carbon atoms, double bonds, methylene, methyl, olefin, alkene, fluorinated, electroactive, conjugated, azido, carbonyl, alkyl, imino, and unsaturated functionalities. 
     
     
         15 . The method of  claim 1 , wherein the polypeptide is selected from the group consisting of insulin, growth hormones, interferons, serum albumins, and epidermal growth factors. 
     
     
         16 . A polypeptide molecule produced by the method of  claim 1 . 
     
     
         17 . The polypeptide of  claim 16 , wherein said polypeptide is further modified by chemical modification of the amino acid analogue. 
     
     
         18 . The polypeptide of  claim 17 , wherein said chemical modification is selected from the group consisting of cycloaddition, substitution, palladium-catalyzed coupling, olefin metathesis, and other chemistries. 
     
     
         19 . The chemical substituent of  claim 17 , comprising site-specific methylation, phosphorylation or addition of sugar chains. 
     
     
         20 . The polypeptide of  claim 16 , which includes insulin, growth hormones, interferons, serum albumin, or epidermal growth factors. 
     
     
         21 . The method of  claim 1 , wherein the amino acid analogue is an analogue with the highest k cat /K m  values so as to support the highest levels of protein synthesis. 
     
     
         22 . The method of  claim 1 , wherein the amino acid analogues is an analogue that has rates of ATP-PPi exchange higher than the corresponding natural amino acid, by the corresponding aminoacyl-tRNA synthetases. 
     
     
         23 . An aminoacyl-tRNA synthetase modified by site-directed mutagenesis and/or directed evolution to enhance properties of the enzyme to facilitate the incorporation of an amino acid analogue into a polypeptide of interest. 
     
     
         24 . The synthetase of  claim 23 , wherein the modification results in improved kinetics of activation of the analogue by the synthetase. 
     
     
         25 . The synthetase of  claim 24 , wherein the kinetics of activation are improved by providing a Km for the amino acid analogue that is lower than the Km for the corresponding natural amino acid. 
     
     
         26 . The synthetase of  claim 24 , wherein the kinetics of activation are improved by providing a Kcat for the amino acid analogue that is higher than the Kcat for the corresponding natural amino acid. 
     
     
         27 . A host cell comprising an aminoacyl-tRNA synthetase modified by site-directed mutagenesis and/or directed evolution to enhance properties of the enzyme to facilitate the incorporation of an amino acid analogue into a polypeptide of interest.