Construction of a bacterial surface display system based on outer membrane protein F
Construction of a bacterial surface display system based on outer membrane protein F
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基于外膜蛋白F的细菌表面展示系统的构建
DOI：
10.1186/s12934-019-1120-2
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发表时间：
2019-04-11
影响因子：
6.4
通讯作者：
Xia, Ningshao
中科院分区：
文献类型：
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作者：
Chen, Tingting;Wang, Kaihang;Xia, Ningshao
背景细菌表面展示系统被开发用于表面展示异源蛋白或多肽，用于不同的应用，如多肽文库筛选和活菌疫苗设计。各种外膜蛋白，如外膜蛋白A(OmpA)、OMPC和外膜孔蛋白E前体(PHOE)已被用作表面展示的载体，与革兰氏阴性菌中感兴趣的蛋白或多肽融合。在这里，我们研究了组成性表达的OmpF在大肠杆菌表面展示外源免疫表位的作用，并将其与质粒诱导的OmpF和OmPC的表达进行了比较。结果OmpF的增强表达与OmpF启动子序列的突变有关。这种突变使OmpF成为理想的载体蛋白，用于在细菌表面丰富展示感兴趣的目标。为此，我们嫁接了两个含有乙肝病毒S抗原和人乳头瘤病毒L2蛋白重要表位的多肽ontoOmpFofE。科利比基因组编辑。融合的OmpF蛋白在编辑的E。膜组分萃取法提纯大肠菌群。通过SDS-PAGE、Western blotting、流式细胞仪和免疫电子显微镜对完整细菌的表位进行了验证。我们使用相同的方法进一步比较了这种结构性表达与质粒诱导的OmpF和OMPC在细菌细胞中的表达。结论以非依赖于质粒的方式增强了OmpF的表达，为在细菌表面展示表位提供了一种顺从的方式，为生物技术工程菌的应用奠定了基础。
BackgroundBacterial surface display systems were developed to surface expose heterologous proteins or peptides for different applications, such as peptide libraries screening and live bacterial vaccine design. Various outer membrane proteins, such as outer membrane protein A (OmpA), OmpC and outer membrane pore protein E precursor (PhoE), have been used as carriers for surface display, fused to the proteins or peptides of interest in Gram-negative bacteria. Here, we investigated the utility of constitutively expressed OmpF for the display of foreign immune epitopes on theEscherichia colicell surface and then compared it with plasmid-induced expression of OmpF and OmpC.ResultsEnhanced expression of OmpF was linked to a mutation in the OmpF promoter sequence. This mutation rendered OmpF an ideal carrier protein for the enriched display of a target of interest on the bacterial surface. To this end, we grafted two peptides, harboring important epitopes of the hepatitis B virus (HBV) S antigen and human papilloma virus (HPV) L2 protein, ontoOmpFofE. coliby genome editing. The resultant fused OmpF proteins were constitutively expressed in the editedE. coliand purified by membrane component extraction. The epitope that displayed on the bacterial surface was verified by SDS-PAGE, western blotting, flow cytometry, and immunoelectron microscopy of the intact bacteria. We further compared this constitutive expression with plasmid-induced expression of OmpF and OmpC in bacterial cells using the same methods for verification. We found that plasmid-induced expression is much less efficient than constitutive expression of OmpF from the bacterial genome.ConclusionsEnhanced expression of OmpF in a plasmid-independent manner provides an amenable way to display epitopes on the bacterial surface and sheds light on ways to engineer bacteria for biotechnological applications.