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%.