Directed protein packaging within outer membrane vesicles from Escherichia coli: Design, production and purification

Nathan Alves, Kendrick B. Turner, Scott A. Walper

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Original languageEnglish (US)
Article numbere54458
JournalJournal of Visualized Experiments
Volume2016
Issue number117
DOIs
StatePublished - Nov 16 2016

Fingerprint

Product Packaging
Escherichia coli
Purification
Packaging
Proteins
Membranes
Recombinant proteins
Recombinant Proteins
Enzymes
Bacteria
Synthetic Biology
Biological Phenomena
Enzyme Stability
Gene transfer
Virulence Factors
Gram-Negative Bacteria
Cell Communication
Pharmaceutical Preparations
Nanoparticles
Anchors

Keywords

  • Biochemistry
  • Bioorthogonal linkage
  • Directed packaging
  • E. coli
  • Enhanced stability
  • Enzyme
  • Outer membrane vesicles (OMV)
  • Phosphotriesterase (PTE)
  • Purification

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Directed protein packaging within outer membrane vesicles from Escherichia coli : Design, production and purification. / Alves, Nathan; Turner, Kendrick B.; Walper, Scott A.

In: Journal of Visualized Experiments, Vol. 2016, No. 117, e54458, 16.11.2016.

Research output: Contribution to journalArticle

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