Recombinant n-glycosylated proteins from procaryotic cells

Abstract

The present invention relates to recombinant N-glycosylated proteins, comprising one or more introduced N-glycosylated optimized amino acid sequence(s), nucleic acids encoding these proteins as well as corresponding vectors and host cells. In addition, the present invention is directed to the use of said proteins, nucleic acids, vectors and host cells for preparing medicaments. Furthermore, the present invention provides methods for producing said proteins.

1 - 27 . (canceled) 
     
     
         28 . A method for producing N-linked glycosylated proteins, comprising:
 a) providing a recombinant prokaryotic organism comprising nucleic acids coding for
 i) a functional pgl operon from  Campylobacter jejuni , and 
 ii) at least one recombinant target protein comprising one or more of the following N-glycosylated optimized amino acid consensus sequence(s): D/E-X-N-Z-S/T, wherein X and Z may be any natural amino acid except Pro, and wherein at least one of said N-glycosylated optimized amino acid consensus sequence(s) is introduced, and 
   b) culturing the recombinant organism in a manner suitable for the production and N-glycosylation of the target protein(s).   
     
     
         29 . The method of  claim 28 , wherein the recombinant prokaryotic organism is selected from the group of bacteria consisting of  Escherichia  ssp.,  Campylobacter  ssp.,  Salmonella  ssp.,  Shigella  ssp.,  Helicobacter  ssp.,  Pseudomonas  ssp., and  Bacillus  ssp. 
     
     
         30 . The method of  claim 28 , wherein the functional pgl operon from  Campylobacter jejuni  comprises nucleic acids coding for
 i) recombinant oligosaccharyl transferase (OTase) from  Campylobacter jejuni , and   ii) recombinant or natural specific glycosyltransferases from   (a)  Campylobacter jejuni , or   (b) species other than  Campylobacter  spp., or   (c) both (a) and (b),   for the assembly of an oligosaccharide on a lipid carrier to be transferred to the target protein by the OTase.   
     
     
         31 . A method for preparing a host cell, comprising the steps of:
 i) providing a Gram-negative bacterium,   ii) introducing into said bacterium at least one nucleotide sequence encoding for
 a) at least one recombinant specific glycosyltransferase for the assembly of an oligosaccharide on a lipid carrier, 
 b) at least one recombinant oligosaccharyl transferase (OTase) from  Campylobacter jejuni , and/or 
 c) at least one recombinant protein comprising one or more of the following N-glycosylated optimized amino acid consensus sequence(s): D/E-X-N-Z-S/T, wherein X and Z may be any natural amino acid except Pro, and wherein at least one of said N-glycosylated optimized amino acid consensus sequence(s) is introduced, and 
   iii) culturing said bacterium until at least one recombinant N-glycosylated protein coded by the nucleotide sequence of c) is located in or on the outer membrane of the Gram-negative bacterium.   
     
     
         32 . The method of  claim 31 , wherein the host cell is selected from the group of bacteria consisting of  Escherichia  ssp.,  Campylobacter  ssp.,  Salmonella  ssp.,  Shigella  ssp.,  Helicobacter  ssp.,  Pseudomonas  ssp., and  Bacillus  ssp. 
     
     
         33 . (canceled)