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.

Figure 1

Current method for the production of conjugate vaccines and in vivo biosynthesis. a: oligosaccharyltransferase PglB, b: carrier protein with signal sequence for secretion to the periplasm, c: undecaprenyl-pyrophosphate-linked polysaccharides.

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).

Figure 3

Growth and glycoconjugate formation in batch culture. AcrA-O1 producing E. coli CLM24 (pMIK44, pGVXN64, pGVXN114) were cultivated in LB medium in a 2 L-bioreactor. The arrow indicates the time point of induction with 2 g L^-1 L-arabinose and 1 mM IPTG. Normalized total cell protein samples were taken at the indicated time points (a to f) and analysed by Western blot using anti-ArcA antibodies.

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).

Figure 5

Fed-batch cultivation of AcrA-O1 producing E. coli with semi-defined glycerol medium using three different feed and induction strategies. Strategy A - filled symbols: Linear feed of glycerol and tryptone from time -3 to 0 h, pulse of IPTG (1 mM) and L-arabinose (2 g L^-1) at time 0 h, linear feed of glycerol, tryptone, L-rabinose (2.75 g L^-1 h^-1) and IPTG (80 μM h^-1) from time 0 to 15 h. Strategy B - open symbols: Pulse of glycerol, tryptone, L-arabinose (4 g L^-1) and IPTG (1 mM) at time 0 h, pulse of glycerol, tryptone and L-arabinose (4 g L^-1) at time 4 h. Strategy C - shaded symbols: Pulse of glycerol, yeast extract and tryptone at time -2.6 h; pulse of glycerol, tryptone, L-arabinose (10 g L^-1) and IPTG (1 mM) at time 0 h; linear feed of tryptone, L-arabinose (1.6 g L^-1 h^-1) and IPTG (10 μM h^-1) from time 0 to 15 h. (A) Logarithmic growth curve (circles) and time course of biomass concentrations (squares), time 0 h (broken line): induction with L-arabinose and IPTG. (B) Time course of AcrA and AcrA-O1 formation. Normalized total cell protein samples were analysed by Western blot with anti-AcrA antibodies, numbers indicate time after induction, SF: samples from LB shake flask cultures. Lane B: same sample of strategy B as in middle blot, analysed on a blot with samples of strategy C (shorter development time, non-relevant lanes removed). E. coli CLM24 (pMIK44, pGVXN64, pGVXN114).

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.