Production of glycoprotein vaccines in Escherichia coli
Open Access
- 11 August 2010
- journal article
- Published by Springer Science and Business Media LLC in Microbial Cell Factories
- Vol. 9 (1), 61
- https://doi.org/10.1186/1475-2859-9-61
Abstract
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. 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. 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.Keywords
This publication has 42 references indexed in Scilit:
- Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overviewMicrobial Cell Factories, 2008
- Rationalizing membrane protein overexpressionTrends in Biotechnology, 2006
- Substrate specificity of bacterial oligosaccharyltransferase suggests a common transfer mechanism for the bacterial and eukaryotic systemsProceedings of the National Academy of Sciences of the United States of America, 2006
- Definition of the bacterial N-glycosylation site consensus sequenceThe EMBO Journal, 2006
- Lactose-induced Production of Human Soluble B Lymphocyte Stimulator (hsBLyS) in E. coli with Different Culture StrategiesBiotechnology Letters, 2006
- Specific Growth Rate Determines the Sensitivity of Escherichia coli to Thermal, UVA, and Solar DisinfectionApplied and Environmental Microbiology, 2006
- A set of compatible tac promoter expression vectorsGene, 1996
- Over-production of Proteins inEscherichia coli: Mutant Hosts that Allow Synthesis of some Membrane Proteins and Globular Proteins at High LevelsJournal of Molecular Biology, 1996
- Large-scale fermentation and purification of a recombinant protein from Escherichia coliProcess Biochemistry, 1991
- Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coliGene, 1988