Production of glycoprotein vaccines in Escherichia coli

Microbial Cell Factories

Research

Sandro Dilettoso¹  Linda Thöny-Meyer¹  Julian Ihssen¹  Michael Kowarik²  Cyril Tanner²  Michael Wacker² 

[1]Empa, Swiss Federal Laboratories for Materials Testing and Research, Laboratory for Biomaterials, Lerchenfeldstrasse 5, CH-901, St. Gallen, Switzerland

[2]GlycoVaxyn AG, Grabenstrasse 3, CH-895, Schlieren, Switzerland

关键词: Specific Growth Rate;    Carrier Protein;    Conjugate Vaccine;    Feed Solution;    Shake Flask Culture;   

DOI  :  10.1186/1475-2859-9-61

 received in 2010-04-23, accepted in 2010-08-11,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundConjugate 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.ResultsTwo 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.ConclusionsThe 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.

【 授权许可】

Unknown   
© Ihssen et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.