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Production of glycoprotein vaccines in Escherichia coli.

Ihssen J, Kowarik M, Dilettoso S, Tanner C, Wacker M, Thöny-Meyer L - Microb. Cell Fact. (2010)

Bottom Line: Conjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens.It was found that efficiency of glycosylation but not carrier protein expression was highly susceptible to the physiological state at induction.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Empa, Swiss Federal Laboratories for Materials Testing and Research, Laboratory for Biomaterials, Gallen, Switzerland.

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.

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Glycosylation of AcrA and EPA with Shigella O1 polysaccharides. Extracts of periplasmic proteins from E. coli CLM24 expressing carrier protein, Shigella polysaccharides (pGVXN64) and either wild-type (wt; pGVXN114) or inactive PglB (mut; pGVXN115) were analysed by Western blot. Lanes 1 and 2: AcrA-expressing strain (pMIK44) analysed with anti-AcrA antibodies; lanes 3 and 4: AcrA-expressing strain analysed with anti-Shigella O1 antibodies (same SDS-polyacrylamide gel as lanes 1 and 2); lanes 5 and 6: EPA-expressing strain (pGVXN150) analysed with anti-EPA antibodies; lanes 7 and 8: EPA-expressing strain analysed with anti-Shigella O1 antibodies (same SDS-polyacrylamide gel as lanes 5 and 6).
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Figure 2: Glycosylation of AcrA and EPA with Shigella O1 polysaccharides. Extracts of periplasmic proteins from E. coli CLM24 expressing carrier protein, Shigella polysaccharides (pGVXN64) and either wild-type (wt; pGVXN114) or inactive PglB (mut; pGVXN115) were analysed by Western blot. Lanes 1 and 2: AcrA-expressing strain (pMIK44) analysed with anti-AcrA antibodies; lanes 3 and 4: AcrA-expressing strain analysed with anti-Shigella O1 antibodies (same SDS-polyacrylamide gel as lanes 1 and 2); lanes 5 and 6: EPA-expressing strain (pGVXN150) analysed with anti-EPA antibodies; lanes 7 and 8: EPA-expressing strain analysed with anti-Shigella O1 antibodies (same SDS-polyacrylamide gel as lanes 5 and 6).

Mentions: In previous studies it was shown that the glycoprotein AcrA, a periplasmic component of a multidrug efflux pump in C. jejuni, was N-glycosylated with E. coli O7, E. coli 9a, E. coli O16, P. aeruginosa O11 and C. jejuni oligo- and polysaccharides in recombinant E. coli when functional oligosaccharyltransferase PglB was co-expressed [8,9]. Here we show that AcrA (40 kDa) was glycosylated with S. dysenteriae type 1 polysaccharides (Shigella O1) in E. coli CLM24 [9] containing plasmids pMIK44 (periplasmic AcrA expression), pGVXN114 (PglB expression) and pGVXN64 (gene cluster for Shigella O1 synthesis) (Figure 2, lane 1). Glycosylation of AcrA with Shigella O1 was abolished when pGVXN114 was replaced by pGVXN115, a plasmid encoding the inactive oligosaccharyltransferase variant PglBmut (Figure 2, lane 2). Antibodies against Shigella O1 antigens reacted with glycosylated AcrA while no glycoprotein was detected in periplasmic extract of the PglBmut strain (Figure 2, lanes 3 and 4). The weak bands around 40 kDa visible in lane 4 may be due to contamination with UDP-linked O antigens.


Production of glycoprotein vaccines in Escherichia coli.

Ihssen J, Kowarik M, Dilettoso S, Tanner C, Wacker M, Thöny-Meyer L - Microb. Cell Fact. (2010)

Glycosylation of AcrA and EPA with Shigella O1 polysaccharides. Extracts of periplasmic proteins from E. coli CLM24 expressing carrier protein, Shigella polysaccharides (pGVXN64) and either wild-type (wt; pGVXN114) or inactive PglB (mut; pGVXN115) were analysed by Western blot. Lanes 1 and 2: AcrA-expressing strain (pMIK44) analysed with anti-AcrA antibodies; lanes 3 and 4: AcrA-expressing strain analysed with anti-Shigella O1 antibodies (same SDS-polyacrylamide gel as lanes 1 and 2); lanes 5 and 6: EPA-expressing strain (pGVXN150) analysed with anti-EPA antibodies; lanes 7 and 8: EPA-expressing strain analysed with anti-Shigella O1 antibodies (same SDS-polyacrylamide gel as lanes 5 and 6).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Glycosylation of AcrA and EPA with Shigella O1 polysaccharides. Extracts of periplasmic proteins from E. coli CLM24 expressing carrier protein, Shigella polysaccharides (pGVXN64) and either wild-type (wt; pGVXN114) or inactive PglB (mut; pGVXN115) were analysed by Western blot. Lanes 1 and 2: AcrA-expressing strain (pMIK44) analysed with anti-AcrA antibodies; lanes 3 and 4: AcrA-expressing strain analysed with anti-Shigella O1 antibodies (same SDS-polyacrylamide gel as lanes 1 and 2); lanes 5 and 6: EPA-expressing strain (pGVXN150) analysed with anti-EPA antibodies; lanes 7 and 8: EPA-expressing strain analysed with anti-Shigella O1 antibodies (same SDS-polyacrylamide gel as lanes 5 and 6).
Mentions: In previous studies it was shown that the glycoprotein AcrA, a periplasmic component of a multidrug efflux pump in C. jejuni, was N-glycosylated with E. coli O7, E. coli 9a, E. coli O16, P. aeruginosa O11 and C. jejuni oligo- and polysaccharides in recombinant E. coli when functional oligosaccharyltransferase PglB was co-expressed [8,9]. Here we show that AcrA (40 kDa) was glycosylated with S. dysenteriae type 1 polysaccharides (Shigella O1) in E. coli CLM24 [9] containing plasmids pMIK44 (periplasmic AcrA expression), pGVXN114 (PglB expression) and pGVXN64 (gene cluster for Shigella O1 synthesis) (Figure 2, lane 1). Glycosylation of AcrA with Shigella O1 was abolished when pGVXN114 was replaced by pGVXN115, a plasmid encoding the inactive oligosaccharyltransferase variant PglBmut (Figure 2, lane 2). Antibodies against Shigella O1 antigens reacted with glycosylated AcrA while no glycoprotein was detected in periplasmic extract of the PglBmut strain (Figure 2, lanes 3 and 4). The weak bands around 40 kDa visible in lane 4 may be due to contamination with UDP-linked O antigens.

Bottom Line: Conjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens.It was found that efficiency of glycosylation but not carrier protein expression was highly susceptible to the physiological state at induction.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Empa, Swiss Federal Laboratories for Materials Testing and Research, Laboratory for Biomaterials, Gallen, Switzerland.

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.

Show MeSH
Related in: MedlinePlus