US2014335127A1PendingUtilityA1

System and method for the production of recombinant glycosylated proteins in a prokaryotic host

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Assignee: ETH ZürichPriority: Mar 7, 2002Filed: Feb 25, 2014Published: Nov 13, 2014
Est. expiryMar 7, 2022(expired)· nominal 20-yr term from priority
Inventors:Markus AebiMichael Wacker
C12P 21/005C07K 2317/92A61K 39/02C07K 16/121A61P 31/00A61K 47/549C12N 9/1048C12N 15/70C12N 9/10C12P 21/00
60
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Claims

Abstract

A system and a method for the production of recombinant N-glycosylated target proteins. The system comprises a prokaryotic organism (e.g. Escherichia coli ) into which is introduced a genetic information encoding for a metabolic apparatus capable of carrying out the requested N-glycosylation of the target protein. Said prokaryotic organism also contains the genetic information required for the expression of one or more recombinant target proteins. The metabolic apparatus preferably comprises specific glycosyltransferases for the assembly of the oligosaccharide on a lipid carrier and an OTase that covalently links this oligosaccharide to specific residues of the desired protein.

Claims

exact text as granted — not AI-modified
1 .- 13 . (canceled) 
     
     
         14 . A prokaryotic host cell comprising (i) a target protein comprising the N-glycosylation consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro, and wherein the target protein can be glycosylated at the Asn residue of the consensus sequence; and (ii) a heterologous oligosaccharyl transferase that links the consensus sequence to an oligo- or polysaccharide, wherein said oligo- or polysaccharide is assembled on undecaprenyl pyrophosphate, and wherein said oligosaccharyl transferase is from  Campylobacter jejuni.    
     
     
         15 . The prokaryotic host cell of  claim 14 , wherein said prokaryotic host cell is  Escherichia coli.    
     
     
         16 . The prokaryotic host cell of  claim 14 , wherein said target protein is a bacterial protein. 
     
     
         17 . The prokaryotic host cell of  claim 15 , wherein said target protein is a bacterial protein. 
     
     
         18 . The prokaryotic host cell of  claim 14 , wherein said prokaryotic host cell is  Escherichia coli  and wherein said target protein is a bacterial protein. 
     
     
         19 . The prokaryotic host cell of  claim 14 , wherein said target protein is heterologous to the host cell. 
     
     
         20 . The prokaryotic host cell of  claim 15 , wherein said target protein is heterologous to the host cell. 
     
     
         21 . The prokaryotic host cell of  claim 19 , wherein said target protein is a mammalian protein. 
     
     
         22 . The prokaryotic host cell of  claim 20 , wherein said target protein is a mammalian protein. 
     
     
         23 . The prokaryotic host cell of  claim 19 , wherein said prokaryotic host cell is  Escherichia coli  and wherein said target protein is a mammalian protein. 
     
     
         24 . A method of producing a recombinant N-glycosylated target protein, the method comprising culturing a prokaryotic host cell under conditions suitable for the production of proteins and isolating the N-glycosylated recombinant target protein from said culture, wherein said host prokaryotic host cell comprises (i) a heterologous target protein comprising the N-glycosylation consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro, and wherein the target protein can be glycosylated at the Asn residue of the consensus sequence; and (ii) an oligosaccharyl transferase that links the consensus sequence to an oligo- or polysaccharide, wherein said oligo- or polysaccharide is assembled on undecaprenyl pyrophosphate, and wherein said oligosaccharyl transferase is from  Campylobacter jejuni.    
     
     
         25 . The method of  claim 24 , wherein said prokaryotic host cell is  Escherichia coli.    
     
     
         26 . The method of  claim 24 , wherein said target protein is a bacterial protein. 
     
     
         27 . The method of  claim 25 , wherein said target protein is a bacterial protein. 
     
     
         28 . The method of  claim 24 , wherein said prokaryotic host cell is  Escherichia coli  and wherein said target protein is a bacterial protein. 
     
     
         29 . The method of  claim 24 , wherein said target protein is heterologous to the host cell. 
     
     
         30 . The method of  claim 25 , wherein said target protein is heterologous to the host cell. 
     
     
         31 . The method of  claim 29 , wherein said target protein is a mammalian protein. 
     
     
         32 . The method of  claim 30 , wherein said target protein is a mammalian protein. 
     
     
         33 . The method of  claim 29 , wherein said prokaryotic host cell is  Escherichia coli  and wherein said target protein is a mammalian protein. 
     
     
         34 . A vaccine produced by the method of  claim 24 .

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