Bacterial Nanobioreactors鈥揇irecting Enzyme Packaging into Bacterial Outer Membrane Vesicles

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• 作者：Nathan J. Alves ; Kendrick B. Turner ; Michael A. Daniele ; Eunkeu Oh ; Igor L. Medintz ; Scott A. Walper
• 刊名：ACS Applied Materials & Interfaces
• 出版年：2015
• 出版时间：November 11, 2015
• 年：2015
• 卷：7
• 期：44
• 页码：24963-24972
• 全文大小：511K
• ISSN：1944-8252

文摘

All bacteria shed outer membrane vesicles (OMVs) loaded with a diverse array of small molecules, proteins, and genetic cargo. In this study we sought to hijack the bacterial cell export pathway to simultaneously produce, package, and release an active enzyme, phosphotriesterase (PTE). To accomplish this goal the SpyCatcher/SpyTag (SC/ST) bioconjugation system was utilized to produce a PTE-SpyCatcher (PTE-SC) fusion protein and a SpyTagged transmembrane porin protein (OmpA-ST), known to be abundant in OMVs. Under a range of physiological conditions the SpyTag and SpyCatcher domains interact with one another and form a covalent isopeptide bond driving packaging of PTE into forming OMVs. The PTE-SC loaded OMVs are characterized for size distribution, number of vesicles produced, cell viability, packaged PTE enzyme kinetics, OMV loading efficiency, and enzyme stability following iterative cycles of freezing and thawing. The PTE-loaded OMVs exhibit native-like enzyme kinetics when assayed with paraoxon as a substrate. PTE is often toxic to expression cultures and has a tendency to lose activity with improper handling. The coexpression of OmpA-ST with PTE-SC, however, greatly improved the overall PTE production levels by mitigating toxicity through exporting of the PTE-SC and greatly enhanced packaged enzyme stability against iterative cycles of freezing and thawing.