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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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Chemostat cultivation (D = 0.1 h-1) of AcrA-O1 producing E. coli using different growth substrates. (A) Time course of optical density after inoculation, bars indicate the period of L-arabinose (ara) co-feed and arrows indicate the time point of the addition of 1 mM IPTG. (B) Glycoconjugate formation in chemostat cultures analysed with anti-AcrA antibodies on Western blot (normalized samples). (C) Expression of pglB in LB chemostat culture compared to batch culture (anti-HA Western blot, normalized samples).
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Figure 4: Chemostat cultivation (D = 0.1 h-1) of AcrA-O1 producing E. coli using different growth substrates. (A) Time course of optical density after inoculation, bars indicate the period of L-arabinose (ara) co-feed and arrows indicate the time point of the addition of 1 mM IPTG. (B) Glycoconjugate formation in chemostat cultures analysed with anti-AcrA antibodies on Western blot (normalized samples). (C) Expression of pglB in LB chemostat culture compared to batch culture (anti-HA Western blot, normalized samples).

Mentions: Chemostats were induced after two volume changes in order to keep the cultivation time before induction as short as possible as a precaution against genetic changes (e.g., plasmid loss). Although this is somewhat lower than the usually recommended 3-5 volume changes, constant OD600 values during the induction period indicated that the cultures had reached a steady state (Figure 4A). In chemostat culture with LB medium (15 g L-1 of organic nutrients) a steady state OD600 of 3.2 was reached (Figure 4A). Steady state optical densities were increased by a factor of three to four in glucose-LB and glycerol-LB chemostats (Figure 4A), in spite of lower total amounts of organic nutrients in the feed medium (13 g L-1). AcrA was produced after the start of an L-arabinose co-feed in chemostat cultures with all three media (Figure 4B, strong, lower bands). This indicates that the cells were growing under carbon- and energy-limited conditions because the PBAD promoter, which controls acrA expression from plasmid pMIK44, is highly sensitive to catabolite repression [31].


Production of glycoprotein vaccines in Escherichia coli.

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

Chemostat cultivation (D = 0.1 h-1) of AcrA-O1 producing E. coli using different growth substrates. (A) Time course of optical density after inoculation, bars indicate the period of L-arabinose (ara) co-feed and arrows indicate the time point of the addition of 1 mM IPTG. (B) Glycoconjugate formation in chemostat cultures analysed with anti-AcrA antibodies on Western blot (normalized samples). (C) Expression of pglB in LB chemostat culture compared to batch culture (anti-HA Western blot, normalized samples).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927510&req=5

Figure 4: Chemostat cultivation (D = 0.1 h-1) of AcrA-O1 producing E. coli using different growth substrates. (A) Time course of optical density after inoculation, bars indicate the period of L-arabinose (ara) co-feed and arrows indicate the time point of the addition of 1 mM IPTG. (B) Glycoconjugate formation in chemostat cultures analysed with anti-AcrA antibodies on Western blot (normalized samples). (C) Expression of pglB in LB chemostat culture compared to batch culture (anti-HA Western blot, normalized samples).
Mentions: Chemostats were induced after two volume changes in order to keep the cultivation time before induction as short as possible as a precaution against genetic changes (e.g., plasmid loss). Although this is somewhat lower than the usually recommended 3-5 volume changes, constant OD600 values during the induction period indicated that the cultures had reached a steady state (Figure 4A). In chemostat culture with LB medium (15 g L-1 of organic nutrients) a steady state OD600 of 3.2 was reached (Figure 4A). Steady state optical densities were increased by a factor of three to four in glucose-LB and glycerol-LB chemostats (Figure 4A), in spite of lower total amounts of organic nutrients in the feed medium (13 g L-1). AcrA was produced after the start of an L-arabinose co-feed in chemostat cultures with all three media (Figure 4B, strong, lower bands). This indicates that the cells were growing under carbon- and energy-limited conditions because the PBAD promoter, which controls acrA expression from plasmid pMIK44, is highly sensitive to catabolite repression [31].

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