title: Bacterial Nanobioreactors--Directing Enzyme Packaging into Bacterial Outer Membrane Vesicles.
abstract: 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.
authors.affiliation: National Research Council, 500 Fifth Street NW (Keck 576), Washington, DC 20001, United States.
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Alves
authors.forename: Kendrick B
authors.initials: KB
authors.lastname: Turner
authors.forename: Michael A
authors.initials: MA
authors.lastname: Daniele
authors.affiliation: Sotera Defense Solution, Inc. 7230 Lee DeForest Drive, Columbia, Maryland 21046, United States.
authors.forename: Eunkeu
authors.initials: E
authors.lastname: Oh
authors.forename: Igor L
authors.initials: IL
authors.lastname: Medintz
authors.forename: Scott A
authors.initials: SA
authors.lastname: Walper
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D019149:Bioreactors; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D005615:Freezing; D008855:Microscopy, Electron, Scanning; D036103:Nanotechnology / Q000379:methods*; D044345:Phosphoric Triester Hydrolases / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004798:Enzymes; D011993:Recombinant Fusion Proteins; C078167:OMPA outer membrane proteins; D044345:Phosphoric Triester Hydrolases
keywords: E. coli; SpyCatcher; SpyTag; directed packaging; enzyme; outer membrane vesicle (OMV); phosphotriesterase (PTE)