Kim Jae-Young, Doody Anne M, Chen David J, Cremona Gina H, Shuler Michael L, Putnam David, DeLisa Matthew P
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
J Mol Biol. 2008 Jun 27;380(1):51-66. doi: 10.1016/j.jmb.2008.03.076. Epub 2008 Apr 9.
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 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.
我们通过将几种异源蛋白与大肠杆菌的囊泡相关毒素ClyA融合，构建出了功能显著增强的细菌外膜囊泡（OMV）。与天然未融合的ClyA相似，嵌合的ClyA融合蛋白定位于细菌OMV中，并保留了融合伙伴的活性，首次证明ClyA可用于将完全功能性的异源蛋白直接共定位于细菌OMV中。例如，ClyA与β-内酰胺酶和有机磷水解酶的融合产生了分别能够水解β-内酰胺抗生素和对氧磷的合成OMV。同样，与ClyA C末端融合的抗地高辛单链Fv抗体片段的表达产生了能够紧密且特异性结合抗体同源抗原的定制“免疫MV”。最后，展示与ClyA C末端融合的绿色荧光蛋白的OMV具有高荧光性，并且由于这种新功能，在囊泡与人类上皮细胞相互作用期间可以很容易地被追踪。我们预计，ClyA作为融合伙伴所表现出的相对可塑性将被证明对以下方面有用：（i）进一步的机制研究，以确定调节OMV分泌的囊泡形成机制，并绘制含ClyA的OMV在宿主细胞入侵期间的细胞内路径；（ii）生物技术应用，如蛋白质的表面展示和生物制剂的递送。
