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.

1 .- 26 . (canceled) 
     
     
         27 . A host cell comprising an aminoacyl-tRNA synthetase modified by site-directed mutagenesis and/or directed evolution to enhance properties of the synthetase to facilitate the incorporation of an amino acid analogue into a polypeptide of interest. 
     
     
         28 . The host cell of  claim 27 , wherein the synthetase modification results in improved kinetics of activation of the analogue by the synthetase. 
     
     
         29 . The host cell of  claim 28 , 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. 
     
     
         30 . A host cell comprising an aminoacyl-tRNA synthetase, wherein said synthetase has a Kcat for the amino acid analogue that is higher than the Kcat for the corresponding natural amino acid. 
     
     
         31 . A host cell comprising an aminoacyl-tRNA synthetase, wherein said synthetase incorporates an amino acid analogue into a polypeptide of interest with at least 77% efficiency as compared to incorporation of the corresponding natural amino acid. 
     
     
         32 . The host cell of  claim 31 , wherein said efficiency of incorporation is at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, or at least 90-100%. 
     
     
         33 . A host cell comprising a first vector having a first polynucleotide sequence encoding an aminoacyl-tRNA synthetase; a second vector having a second polynucleotide sequence encoding a polypeptide; and the polypeptide having incorporated therein an amino acid analogue; wherein the first and second vectors may be the same or different, and wherein the amino acid analogue is incorporated into the polypeptide with an efficiency of incorporation of at least 77% as compared to the incorporation of a corresponding natural amino acid. 
     
     
         34 . The host cell of  claim 33  wherein the efficiency of incorporation is at least 90±6% or at least 90-100%. 
     
     
         35 . The host of  claim 34  wherein the efficiency of incorporation is at least 85%, 86%, 87%, 85-90%, 91±2%, 96±2%, 98±2%, 90-96%, or 92-98%. 
     
     
         36 . The host cell of  claim 33  wherein the aminoacyl-tRNA synthetase is from a eukaryotic cell or a prokaryotic cell. 
     
     
         37 . The host cell of  claim 33  wherein the aminoacyl-tRNA synthetase is natural, synthetic, semi-synthetic or recombinant. 
     
     
         38 . The host cell of  claim 37  wherein the recombinant aminoacyl-tRNA synthetase is mutated or genetically engineered. 
     
     
         39 . The host cell of  claim 38  wherein the aminoacyl-tRNA synthetase recognizes the amino acid analogue and is able to facilitate the incorporation of the amino acid analogue into the polypeptide. 
     
     
         40 . The host cell of  claim 33  wherein the amino acid analogue differs from the corresponding natural amino acid by having fluorinated, electroactive, conjugated, azido, carbonyl, alkyl, modifications of the natural amino acid in the side chain functionality, or modifications of the natural amino acid. 
     
     
         41 . The host cell of  claim 40  wherein the amino acid analogues 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 natural amino acids with sides chains containing divalent non-carbon atoms, double bonds, methylene, methyl, olefin, or alkene. 
     
     
         42 . The host cell of  claim 33  wherein the polypeptide is natural, synthetic, semi-synthetic or recombinant. 
     
     
         43 . The host cell of  claim 33  wherein the polypeptide is a hormone, an enzyme, or a protein fiber. 
     
     
         44 . The host cell of  claim 43  wherein the polypeptide is an interferon, a growth factor, a serum albumin, or an epidermal growth factor. 
     
     
         45 . The host cell of  claim 44  wherein the viral DNA is vaccinia, adenovirus, foul pox virus, pseudorabies, or a derivative of SV40. 
     
     
         46 . The host cell of  claim 33  wherein the first and second vector comprises at least one expression element. 
     
     
         47 . The host cell of  claim 46 , 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. 
     
     
         48 . The host cell of  claim 33  wherein host cell is prokaryotic or eukaryotic. 
     
     
         49 . The host cell of  claim 48  wherein the prokaryotic host cell is a bacterial cell. 
     
     
         50 . The host cell of  claim 49  wherein the bacterial cell is a strain of  Escherichia, Bacillus, Pseudomonas, Streptococcus , or  Streptomyces.    
     
     
         51 . The host cell of  claim 48  wherein the eukaryotic host cell is a yeast cell, an insect cell, a plant cell, or an animal cell. 
     
     
         52 . The host cell of  claim 51  wherein the animal cell is a mammalian cell. 
     
     
         53 . The host cell of  claim 52  wherein the mammalian cell is CHO, COS or Bowes melanoma. 
     
     
         54 . The host cell of  claim 33  wherein the host cell is an auxotroph.