In vivo site-sepecific incorporation of N-acetyl-galactosamine amino acids in eubacteria

Abstract

Methods and compositions for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid having a N-acetylgalactosamine moiety into a protein; optionally, the N-acetylgalactosamine-containing unnatural amino acid can be further modified with additional sugars.

1  to  22 . (canceled) 
     
     
         23 . A translation system comprising:
 (a) an unnatural amino acid comprising an N-acetylgalactosamine moiety;   (b) an orthogonal aminoacyl-tRNA synthetase (O—RS); and   (c) an orthogonal tRNA (O-tRNA);   wherein said O—RS comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5.   
     
     
         24 . The translation system of  claim 23 , wherein said unnatural amino acid is selected from the group consisting of: an N-acetylgalactosamine-α-threonine, a triacetyl-N-acetylgalactosamine-α-threonine, an N-acetylgalactosamine-α-serine, and a triacetyl-N-acetylgalactosamine-α-serine. 
     
     
         25 . The translation system of  claim 23 , wherein said O—RS is derived from a wild-type  Methanococcus jannaschii  tyrosyl-tRNA synthetase. 
     
     
         26 . The translation system of  claim 23 , wherein said O—RS comprises an amino acid sequence selected from the amino acids sequences set forth in SEQ ID NOs: 1, 2, 3, or 4. 
     
     
         27 . The translation system of  claim 23 , wherein said O-tRNA is an amber suppressor tRNA. 
     
     
         28 . The translation system of  claim 23 , further comprising a nucleic acid encoding a protein of interest, said nucleic acid comprising at least one selector codon, wherein said selector codon is recognized by said O-tRNA. 
     
     
         29 . The translation system of  claim 23 , wherein said translation system comprises a host cell wherein said host cell comprises said unnatural amino acid, said O—RS and said O-tRNA. 
     
     
         30 . The translation system of  claim 29 , wherein said host cell is an  E. coli  cell. 
     
     
         31 . The translation system of  claim 29 , wherein said host cell comprises a polynucleotide encoding said O—RS. 
     
     
         32 . The translation system of  claim 31 , wherein said polynucleotide comprises a nucleotide sequence set forth in SEQ ID NOs: 6, 7, 8 or 9. 
     
     
         33 . The translation system of  claim 29 , wherein said host cell comprises a polynucleotide encoding said O-tRNA.