Engineered bacterial outer membrane vesicles with enhanced functionality
Engineered bacterial outer membrane vesicles with enhanced functionality
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DOI：
10.1016/j.jmb.2008.03.076
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发表时间：
2008-06-27
影响因子：
5.6
通讯作者：
DeLisa, Matthew P.
中科院分区：
文献类型：
--
作者：
Kim, Jae-Young;Doody, Anne M.;DeLisa, Matthew P.
我们已经设计了细菌外膜囊泡（OMV），通过融合几种异源蛋白到大肠杆菌的囊泡相关毒素ClyA，具有显著增强的功能。与天然未融合的ClyA类似，发现嵌合ClyA融合蛋白定位于细菌OMV中并保留融合配偶体的活性，首次证明ClyA可用于直接在细菌OMV中共定位完全功能的异源蛋白和酶。例如，ClyA与β-内酰胺酶和有机磷水解酶的融合产生了能够分别水解β-内酰胺抗生素和对氧磷的合成OMV。类似地，与ClyA的C末端融合的抗地高辛单链Fv抗体片段的表达导致设计者“免疫MV”，其可以紧密且特异性地结合抗体的同源抗原。最后，展示融合到ClyA的C末端的绿色荧光蛋白的OMV是高度荧光的，并且由于这种新的功能性，在囊泡与人上皮细胞的相互作用期间可以容易地跟踪。我们预期ClyA作为融合伴侣所表现出的相对可塑性应证明可用于：（i）进一步的机制研究，以鉴定调节OMV分泌的囊泡形成机制，并绘制含有ClyA的OMV在侵入宿主细胞期间的细胞内路线;和（ii）生物技术应用，如蛋白质的表面展示和生物制剂的递送。(C)2008爱思唯尔有限公司保留所有权利。
We have engineered bacterial outer membrane vesicles (OMVs) with dramatically enhanced functionality by fusing several heterologous proteins to the vesicle-associated toxin ClyA of Escherichia coli. Similar to native unfused ClyA, chimeric ClyA fusion proteins were found localized in bacterial OMVs and retained activity of the fusion partners, demonstrating for the first time that ClyA can be used to co-localize fully functional heterologous proteins h e enzymes directly in bacterial OMVs. For instance, fusions of ClyA to the enzymes beta-lactamase and organophosphorus hydrolase resulted in synthetic OMVs that were capable of hydrolyzing beta-lactam antibiotics and paraoxon, respectively. Similarly, expression of an anti-digoxin single-chain Fv antibody fragment fused to the C terminus of ClyA resulted in designer "immuno-MVs" that could bind tightly and specifically to the antibody's cognate antigen. Finally, OMVs displaying green fluorescent protein fused to the C terminus of ClyA were highly fluorescent and, as a result of this new functionality, could be easily tracked during vesicle interaction with human epithelial cells. We expect that the relative plasticity exhibited by ClyA as a fusion partner should prove useful for: (i) further mechanistic studies to identify the vesiculation machinery that regulates OMV secretion and to map the intracellular routing of ClyA-containing OMVs during invasion of host cells; and (ii) biotechnology applications such as surface display of proteins and delivery of biologics. (C) 2008 Elsevier Ltd. All rights reserved.