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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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Production of EPA-O1 in fed-batch culture. Cultivation and induction were performed according to strategy C as described in Materials and methods and legend to Figure 5. Strain: E. coli CLM24 (pGVXN150, pGVXN64, pGVXN114). (A) Logarithmic growth curve (circles) and time course of biomass concentrations (squares). Filled symbols, dotted line: fed-batch run 1 (FB1); open symbols, solid line: fed-batch run 2 (FB2). Time 0 h (broken, vertical line): induction with L-arabinose and IPTG. (B) Time course of EPA and EPA-O1 formation in fed-batch culture compared to LB shake flask culture (SF); anti-EPA Western blots, normalized samples, numbers indicate time after induction.
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Figure 6: Production of EPA-O1 in fed-batch culture. Cultivation and induction were performed according to strategy C as described in Materials and methods and legend to Figure 5. Strain: E. coli CLM24 (pGVXN150, pGVXN64, pGVXN114). (A) Logarithmic growth curve (circles) and time course of biomass concentrations (squares). Filled symbols, dotted line: fed-batch run 1 (FB1); open symbols, solid line: fed-batch run 2 (FB2). Time 0 h (broken, vertical line): induction with L-arabinose and IPTG. (B) Time course of EPA and EPA-O1 formation in fed-batch culture compared to LB shake flask culture (SF); anti-EPA Western blots, normalized samples, numbers indicate time after induction.

Mentions: Fed-batch strategy C was then tested for production of the second glycoconjugate EPA-O1. Two independent fed-batch runs yielded similar levels of glycoprotein as found in LB shake flask cultures after a total cultivation time of 25 h (Figures 6A and 6B). Final OD600 was increased from 2 in shake flask to 80 in fed-batch culture (Table 1), thus volumetric productivity was increased by a factor of 40. Glycoprotein EPA-O1 formation in fed-batch culture was seen with a more pronounced retardation compared to cultures producing AcrA-O1 (Figures 5B and 6B). This difference was not due to reduced or delayed expression of the carrier protein (Bands 40 kDa in Figure 5B and bands 70 kDa in Figure 6B). Similarly to batch cultures, induction of carrier protein and PglB synthesis in fed-batch cultures lead to an immediate reduction of the specific growth rate (Figures 5A and 6A, Table 1).


Production of glycoprotein vaccines in Escherichia coli.

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

Production of EPA-O1 in fed-batch culture. Cultivation and induction were performed according to strategy C as described in Materials and methods and legend to Figure 5. Strain: E. coli CLM24 (pGVXN150, pGVXN64, pGVXN114). (A) Logarithmic growth curve (circles) and time course of biomass concentrations (squares). Filled symbols, dotted line: fed-batch run 1 (FB1); open symbols, solid line: fed-batch run 2 (FB2). Time 0 h (broken, vertical line): induction with L-arabinose and IPTG. (B) Time course of EPA and EPA-O1 formation in fed-batch culture compared to LB shake flask culture (SF); anti-EPA Western blots, normalized samples, numbers indicate time after induction.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Production of EPA-O1 in fed-batch culture. Cultivation and induction were performed according to strategy C as described in Materials and methods and legend to Figure 5. Strain: E. coli CLM24 (pGVXN150, pGVXN64, pGVXN114). (A) Logarithmic growth curve (circles) and time course of biomass concentrations (squares). Filled symbols, dotted line: fed-batch run 1 (FB1); open symbols, solid line: fed-batch run 2 (FB2). Time 0 h (broken, vertical line): induction with L-arabinose and IPTG. (B) Time course of EPA and EPA-O1 formation in fed-batch culture compared to LB shake flask culture (SF); anti-EPA Western blots, normalized samples, numbers indicate time after induction.
Mentions: Fed-batch strategy C was then tested for production of the second glycoconjugate EPA-O1. Two independent fed-batch runs yielded similar levels of glycoprotein as found in LB shake flask cultures after a total cultivation time of 25 h (Figures 6A and 6B). Final OD600 was increased from 2 in shake flask to 80 in fed-batch culture (Table 1), thus volumetric productivity was increased by a factor of 40. Glycoprotein EPA-O1 formation in fed-batch culture was seen with a more pronounced retardation compared to cultures producing AcrA-O1 (Figures 5B and 6B). This difference was not due to reduced or delayed expression of the carrier protein (Bands 40 kDa in Figure 5B and bands 70 kDa in Figure 6B). Similarly to batch cultures, induction of carrier protein and PglB synthesis in fed-batch cultures lead to an immediate reduction of the specific growth rate (Figures 5A and 6A, Table 1).

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