Production of glycoprotein vaccines in Escherichia coli

Abstract

BackgroundConjugate 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.ResultsTwo 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.ConclusionsThe 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.

The Sweet Tooth of Bacteria: Common Themes in Bacterial Glycoconjugates

“…This technology is extremely interesting for industrial applications such as the amelioration of food quality by the engineering of EPS structures or the production of engineered glycoproteins, such as vaccines and human therapeutic agents (e.g., insulin) (131,(559)(560)(561)(562)(563)(564)(565)(566)(567)(568). A better knowledge of the glycosylation mechanisms in beneficial bacteria could cause a revolution in the biosynthesis of therapeutic molecules in prokaryotic vectors.…”

Characterization of the Structurally Diverse N-Linked Glycans of Campylobacter Species

“…The extensively characterized C. jejuni N-linked protein glycosylation system is now established as both a model for understanding protein glycosylation and a biotechnological tool for in vivo generation of glycoconjugates (5,12,14,50). However, genome sequence data indicate that not only C. jejuni but all Campylobacter species contain a gene cluster encoding a putative N-linked protein glycosylation system.…”

“…When structurally variant lipid-linked glycans are made available, the C. jejuni PglB OTase is able to transfer many, but not all, of these onto proteins (5,49). Such flexibility makes this an attractive approach for the development of an in vivo glycoconjugate synthesis system (14). The key structural requirement for PglB-mediated glycan transfer is the presence of an acetamido group on the C-2 carbon of the reducingend sugar (5,49).…”

Fabrication of nanocomposites and hybrid materials using microbial biotemplates

“…On the other hand, metabolites produced in microbial cell factories such as small Fig. 4 Two approaches to creating synthetic microbes: (1) creating life de novo, creating an artificial cell at least needs to integrate three main components: containment, cellular processes, and information and (2) bioengineering approach, using a refined version of genetic engineering, designing, and inserting elements inside the cells to perform specialized functions molecule (e.g., ethanol [89], lactic acid [90,91], glycerol [92]), antibiotic [93] (e.g., penicillin), pigment [94], protein [95] and glycoprotein [96], polysaccharide (e.g., hyaluronic acid [97], bacterial cellulose [98]), and even virus-like particles (as vaccine) [99] have been widely used in the production and life of human beings (Fig. 6).…”