Browsing by Subject "outer membrane vesicles"

Now showing 1 - 3 of 3
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    Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant
    (Elsevier, 2017-02) Alves, Nathan J.; Turner, Kendrick B.; DiVito, Kyle A.; Daniele, Michael A.; Walper, Scott A.; Department of Emergency Medicine, School of Medicine
    To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function.
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    Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification
    (2016) Alves, Nathan J.; Turner, Kendrick B.; Walper, Scott A.; Department of Emergency Medicine, School of Medicine
    A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.
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    Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles
    (Nature, 2016-04) Alves, Nathan J.; Turner, Kendrick B.; Medintz, Igor L.; Walper, Scott A.; Emergency Medicine, School of Medicine
    Bacteria possess innate machinery to transport extracellular cargo between cells as well as package virulence factors to infect host cells by secreting outer membrane vesicles (OMVs) that contain small molecules, proteins, and genetic material. These robust proteoliposomes have evolved naturally to be resistant to degradation and provide a supportive environment to extend the activity of encapsulated cargo. In this study, we sought to exploit bacterial OMV formation to package and maintain the activity of an enzyme, phosphotriesterase (PTE), under challenging storage conditions encountered for real world applications. Here we show that OMV packaged PTE maintains activity over free PTE when subjected to elevated temperatures (>100-fold more activity after 14 days at 37 °C), iterative freeze-thaw cycles (3.4-fold post four-cycles), and lyophilization (43-fold). We also demonstrate how lyophilized OMV packaged PTE can be utilized as a cell free reagent for long term environmental remediation of pesticide/chemical warfare contaminated areas.