title: Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant.
abstract: 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.
authors: National Research Council, 500 Fifth Street NW, Keck 576, Washington, DC 20001, USA; Department of Emergency Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
authors: Nathan J
authors: NJ
authors: Alves
authors: Center for Bio/Molecular Science & Engineering, Naval Research Laboratory, Washington, DC 20375, USA.
authors: Kendrick B
authors: KB
authors: Turner
authors: American Society for Engineering Education (ASEE), 1818 N Street NW, Suite 600, Washington, DC 20036, USA.
authors: Kyle A
authors: KA
authors: DiVito
authors: North Carolina State University, Joint Department of Biomedical Engineering, UNC-Chapel Hill/NC State University, 2068 Engineering Building 2, Campus Box 7911, Raleigh, NC 27695, USA.
authors: Michael A
authors: MA
authors: Daniele
authors: Center for Bio/Molecular Science & Engineering, Naval Research Laboratory, Washington, DC 20375, USA. Electronic address: Scott.walper@nrl.navy.mil.
authors: Scott A
authors: SA
authors: Walper
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development / Q000648:ultrastructure*; D000067128:Extracellular Vesicles / Q000737:chemistry; D005818:Genetic Engineering / Q000379:methods*; D006639:Histidine / Q000235:genetics; D007763:Lac Operon; D009842:Oligopeptides / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification
chemical_list: D001425:Bacterial Outer Membrane Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; C078167:OMPA outer membrane proteins; D006639:Histidine
keywords: Affinity purification; Escherichia coli; Extracellular vesicles; His-tag; IMAC; Outer membrane vesicles (OMVs)