In vivo production of a novel glycoconjugate vaccine against Shigella flexneri 2a in recombinant Escherichia coli: identification of stimulating factors for in vivo glycosylation.
In vivo production of a novel glycoconjugate vaccine against Shigella flexneri 2a in recombinant Escherichia coli: identification of stimulating factors for in vivo glycosylation.
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DOI：
10.1186/s12934-015-0195-7
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发表时间：
2015-01-23
影响因子：
6.4
通讯作者：
Ren Q
中科院分区：
文献类型：
--
作者：
Kämpf MM;Braun M;Sirena D;Ihssen J;Thöny-Meyer L;Ren Q
由多糖抗原与免疫原性载体蛋白共价连接而成的糖缀合疫苗已被证明属于对抗细菌病原体的最有效和最安全的疫苗。空肠弯曲菌的N-糖基化机制的功能转移到标准的原核宿主大肠杆菌建立了一种新的生物缀合方法，称为细菌糖工程。在这项研究中，我们报告了一种新的重组糖缀合物疫苗的生产对志贺菌福氏2a代表志贺菌病的全球爆发的主要血清型。我们证明了S.弗氏2a O-多糖可通过C.空肠寡糖基转移酶PglB，导致糖基化EPA-2a。此外，我们通过鉴定和定量分析糖蛋白合成的关键工艺参数，优化了这种新型疫苗的体内生产。发现寡糖基转移酶PglB和载体蛋白EPA的顺序诱导使EPA-2a的比生产率增加了1.6倍。此外，通过在诱导期间添加10 g/L的单糖N-乙酰葡糖胺，糖缀合物疫苗产量提高至3.1倍。确定用于N-聚糖转移的Mg 2+离子的最佳浓度为10 mM。最后，将优化的参数转移到高细胞密度培养物中，与初始摇瓶生产相比，糖缀合物的总产率增加了46倍。本研究是首次尝试确定刺激参数，提高生产力的S。弗氏2a生物缀合物。糖基化效率的优化将最终促进实验室规模的表达转移到针对S的糖缀合物疫苗的具有成本效益的体内生产过程。flexneri 2a在E.杆菌本研究是实现这一目标的重要一步，并为进一步的优化研究提供了一个起点。
Glycoconjugated vaccines composed of polysaccharide antigens covalently linked to immunogenic carrier proteins have proved to belong to the most effective and safest vaccines for combating bacterial pathogens. The functional transfer of the N-glycosylation machinery from Campylobacter jejuni to the standard prokaryotic host Escherichia coli established a novel bioconjugation methodology termed bacterial glycoengineering. In this study, we report on the production of a new recombinant glycoconjugate vaccine against Shigella flexneri 2a representing the major serotype for global outbreaks of shigellosis. We demonstrate that S. flexneri 2a O-polysaccharides can be transferred to a detoxified variant of Pseudomonas aeruginosa carrier protein exotoxin A (EPA) by the C. jejuni oligosaccharyltransferase PglB, resulting in glycosylated EPA-2a. Moreover, we optimized the in vivo production of this novel vaccine by identification and quantitative analysis of critical process parameters for glycoprotein synthesis. It was found that sequential induction of oligosaccharyltransferase PglB and carrier protein EPA increased the specific productivity of EPA-2a by a factor of 1.6. Furthermore, by the addition of 10 g/L of the monosaccharide N-acetylglucosamine during induction, glycoconjugate vaccine yield was boosted up to 3.1-fold. The optimum concentration of Mg2+ ions for N-glycan transfer was determined to be 10 mM. Finally, optimized parameters were transferred to high cell density cultures with a 46-fold increase of overall yield of glycoconjugate compared to the one in initial shake flask production. The present study is the first attempt to identify stimulating parameters for improved productivity of S. flexneri 2a bioconjugates. Optimization of glycosylation efficiency will ultimately foster the transfer of lab-scale expression to a cost-effective in vivo production process for a glycoconjugate vaccine against S. flexneri 2a in E. coli. This study is an important step towards this goal and provides a starting point for further optimization studies.
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影响因子：
6.4
作者：
Iwashkiw JA;Fentabil MA;Faridmoayer A;Mills DC;Peppler M;Czibener C;Ciocchini AE;Comerci DJ;Ugalde JE;Feldman MF
通讯作者：
Feldman MF
影响因子：
3.1
作者：
Cohen, D;Ashkenazi, S;Robbins, JB
通讯作者：
Robbins, JB
影响因子：
3.9
作者：
Lecina, Marti;Sarro, Enric;Cairo, Jordi J.
通讯作者：
Cairo, Jordi J.
影响因子：
3.5
作者：
Ihssen, Julian;Kowarik, Michael;Thoeny-Meyer, Linda
通讯作者：
Thoeny-Meyer, Linda
影响因子：
6.4
作者：
Collins T;Azevedo-Silva J;da Costa A;Branca F;Machado R;Casal M
通讯作者：
Casal M