title: Production of glycoprotein vaccines in Escherichia coli.
abstract: BACKGROUND
  Conjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens. State-of-the art production of conjugate vaccines using chemical methods is a laborious, multi-step process. In vivo enzymatic coupling using the general glycosylation pathway of Campylobacter jejuni in recombinant Escherichia coli has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the in vivo biosynthesis of two novel conjugate vaccine candidates against Shigella dysenteriae type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries.
  
  
  RESULTS
  Two different periplasmic carrier proteins, AcrA from C. jejuni and a toxoid form of Pseudomonas aeruginosa exotoxin were glycosylated with Shigella O antigens in E. coli. Starting from shake flask cultivation in standard complex medium a lab-scale fed-batch process was developed for glycoconjugate production. It was found that efficiency of glycosylation but not carrier protein expression was highly susceptible to the physiological state at induction. After induction glycoconjugates generally appeared later than unglycosylated carrier protein, suggesting that glycosylation was the rate-limiting step for synthesis of conjugate vaccines in E. coli. Glycoconjugate synthesis, in particular expression of oligosaccharyltransferase PglB, strongly inhibited growth of E. coli cells after induction, making it necessary to separate biomass growth and recombinant protein expression phases. With a simple pulse and linear feed strategy and the use of semi-defined glycerol medium, volumetric glycoconjugate yield was increased 30 to 50-fold.
  
  
  CONCLUSIONS
  The presented data demonstrate that glycosylated proteins can be produced in recombinant E. coli at a larger scale. The described methodologies constitute an important step towards cost-effective in vivo production of conjugate vaccines, which in future may be used for combating severe infectious diseases, particularly in developing countries.
authors.affiliation: Empa, Swiss Federal Laboratories for Materials Testing and Research, Laboratory for Biomaterials, Gallen, Switzerland.
authors.forename: Julian
authors.initials: J
authors.lastname: Ihssen
authors.forename: Michael
authors.initials: M
authors.lastname: Kowarik
authors.forename: Sandro
authors.initials: S
authors.lastname: Dilettoso
authors.forename: Cyril
authors.initials: C
authors.lastname: Tanner
authors.forename: Michael
authors.initials: M
authors.lastname: Wacker
authors.forename: Linda
authors.initials: L
authors.lastname: Thöny-Meyer
mesh_terms: D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins; D006031:Glycosylation; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D019081:O Antigens / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D022281:Shigella Vaccines / Q000096:biosynthesis*; D012762:Shigella dysenteriae / Q000276:immunology*; D018074:Vaccines, Conjugate / Q000096:biosynthesis
chemical_list: C083497:AcrA protein, E coli; D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D006023:Glycoproteins; D008074:Lipoproteins; D026901:Membrane Transport Proteins; D019081:O Antigens; D011993:Recombinant Fusion Proteins; D022281:Shigella Vaccines; D018074:Vaccines, Conjugate