JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biochemistry

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

Published: November 16th, 2016

DOI:

10.3791/54458

1Center for Bio/Molecular Science & Engineering, Naval Research Laboratory, 2Department of Emergency Medicine, Indiana University of 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.

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.

Presented here is a method for the design, production, and purification of enzyme-loaded bacterial outer membrane vesicles (OMV). OMV are small, primarily unilamellar, proteoliposomes that range in size from 30-200 nm1,2. All Gram-negative and Gram-positive bacteria that have been studied to date have demonstrated release of either OMV or extracellular vesicles (EV) from their surface3,4. The precise mechanism by which OMV are produced have yet to be fully elucidated due to the diverse bacterial populations that secrete them as well as the varying functions that they serve. OMV have been shown to transport a wide range of cargo from small molecul....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Preparation of Plasmids

  1. Prepare a plasmid (e.g., pET22) containing the anchor protein (OmpA) fused to a biorthogonal linkage domain (referred to in this protocol as anchor-ST), epitope tag (such as 6xHis, myc or FLAG tags for purification and identification), periplasmic localization tag, antibiotic resistance, and appropriate origin of replication based on the selected bacterial strain7.
    1. Extract plasmid DNA from overnight cultures using a commercially available DNA isolatio.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Simultaneous expression of two recombinant proteins, as is required for the OMV packaging strategy detailed in this protocol, can be accomplished through a number of different avenues. Here, a two vector system was utilized with compatible origins of replication and separate inducible gene cassettes. For the expression of the PTE-SC construct a commercial plasmid backbone (pACY184) was engineered to include an arabinose inducible gene cassette and a twin arginine periplasmic localization .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This protocol functions to demonstrate a representative directed packaging technique in which an enzyme of interest is produced and packaged into OMV by E. coli. As with many complex techniques there are multiple areas in which the protocol can be modified to accommodate for use in different unique applications, some of which are detailed below. While the mechanism of OMV packaging and enzyme encapsulation can be adapted to specific needs there are several steps within this protocol which are critical to its suc.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This research was funded by the Office of Naval Research through Core funds provided to the Naval Research Laboratory.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
IPTG Any Always prepare fresh or aliquot and freeze.
L-arabinose Any Can be prepared ahead of time and stored at 4C.
Ampicillin Any Add immediately prior to use after media cools sufficiently from being autoclaved.  
Chloramphenicol  Any Add immediately prior to use after media cools sufficiently from being autoclaved.  
TB/LB Culture Media Any Other growth medias will likely work similarly.
Triton X-100 Any One of many potential suitable surfactants.  
Baffled culture flasks Any The baffles promote higher levels of aeration.  
CHES Fisher Bioreagents BP318-100 Optimal buffer used for paraoxon degredation (pH > 8).
Paraoxon Chem Service N-12816 Very toxic substance to be handled carefully and disposed of properly.  
Syringe Filter 0.45 µm Thermo Scientific 60183-221       (30 mm) Filter diameter will depend on volume of sample.  Low protein binding membrane is critical.
Shaker incubator New Brunswick Excella E24 Precise temperature and mixing is essential for reproducable bacterial growth. 
Sorvall Culture Centrifuge Thermo Scientific RC 5B PLUS Large volume (500 mL) culture centrifuge capable of 7,000 x g.
Sorvall Ultracentrifuge  Thermo Scientific WX Ultra 90 Capable of centrifugal forces ≥150,000 x g.
Ultracentrifuge Rotor Thermo Scientific AH-629 Ensure the proper rotor and tubes are used and that everything is properly balanced.
Ultra-Clear Ultracentrifuge Tubes        (25 x 89 mm) Beckman Coulter 344058 Ensure no stress fractures are present prior to use and that tubes are presicely balanced. 
Spectrophotometer  Tecan Infinite M1000 Necessary for enzyme kinetic assays.
DLS / particle tracking NanoSight  LM10 Necessary for OMV size distribution and concentration determination.  
BL21(DE3) NEB Suitable bacterial expression strain.
pET22 EMD Millipore 69744-3 Other plasmids can be used in place of these.  
pACYC184 NEB Other plasmids can be used in place of these.  
Gel Extraction Kit Qiagen 28704 Example kit.

  1. Avila-Calderon, E. D., et al. Roles of bacterial membrane vesicles. Arch. Microbiol. 197, 1-10 (2015).
  2. Kulp, A., Kuehn, M. J. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu. Rev. Microbiol. 64, 163-184 (2010).
  3. Beveridge, T. Structures of Gram negative cell walls and their derived membrane vesicles. J. Bacteriol. 181, 4725-4733 (1999).
  4. Kulkarni, H. M., Jagannadham, M. V. Biogenesis and multifaceted roles of outer membrane vesicles from Gram-negative bacteria. Microbiology. 160, 2109-2121 (2014).
  5. Ellis, T. N., Kuehn, M. J. Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiol. Mol. Biol. Rev. 74, 81-94 (2010).
  6. Berleman, J., Auer, M. The role of bacterial outer membrane vesicles for intra- and interspecies delivery. Environ. Microbiol. 15, 347-354 (2013).
  7. Alves, N. J., et al. Bacterial nanobioreactors-directing enzyme packaging into bacterial outer membrane vesicles. ACS Appl. Mater. Interfaces. 7, 24963-24972 (2015).
  8. Kim, J. Y., et al. Engineered bacterial outer membrane vesicles with enhanced functionality. J. Mol. Biol. 380, 51-66 (2008).
  9. Haurat, M. F., et al. Selective sorting of cargo proteins into bacterial membrane vesicles. J. Biol. Chem. 286, 1269-1276 (2011).
  10. Kesty, N. C., Kuehn, M. J. Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles. J. Biol. Chem. 279, 2069-2076 (2003).
  11. Zakeri, B., et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesion. Proc. Natl. Acad. Sci. U. S. A. 109, e690-e697 (2012).
  12. Li, L., Fierer, J. O., Rapoport, T. A., Howarth, M. Structural analysis and optimization of the covalent association between SpyCatcher and a peptide tag. J. Mol. Biol. 426, 309-317 (2014).
  13. Dumas, D. P., Durst, H. D., Landis, W. G., Raushel, F. M., Wild, J. R. Inactivation of organophosphorus nerve agents by the phophotriesterase from Pseudomonas-Diminuta. Arch. Biochem. Biophys. 277, 155-159 (1990).
  14. Bigley, A. N., Raushel, F. M. Catalytic mechanisms for phosphotriesterases. Biochimica et Biochim. Biophys. Acta, Proteins Proteomics. 1834, 443-453 (2013).
  15. Minton, N. A., Murray, V. S. G. A review of organo-phosphate poisoning. Med. Toxicol. Adverse Drug Exper. 3, 350-375 (1988).
  16. Bigley, A. N., Xu, C., Henderson, T. J., Harvey, S. P., Raushel, F. M. Enzymatic neutralization of the chemical warfare agent VX: Evolution of phosphotriesterase for phosphorothiolate hydrolysis. J. Am. Chem. Soc. 135, 10426-10432 (2013).
  17. Chatterjee, S. N., Chaudhuri, K. Gram-negative bacteria: the cell membranes. Outer Membrane Vesicles of Bacteria. SpringerBriefs in Microbiology. , 15-34 (2012).
  18. Wang, Y. The function of OmpA in Escherichia coli. Biochem. Biophys. Res. Commun. 292, 396-401 (2002).
  19. Danoff, E. J., Fleming, K. G. The soluble, periplasmic domain of OmpA folds as an independent unit and displays chaperone activity by reducing the self-association propensity of the unfolded OmpA transmembrane beta-barrel. Biophys. Chem. 159, 194-204 (2011).
  20. Alves, N. J., Kline, J. A. Comparative study on the inhibition of plasmin and delta-plasmin via benzamidine derivatives. Biochem. Biophys. Res. Commun. 457, 358-362 (2015).
  21. Wang, W. Lyophilization and development of solid protein pharmaceuticals. Int. J. Pharm. 203, 1-60 (2000).
  22. Goormaghtigh, E., Scarborough, G. A. Density-based separation of liposomes by glycerol gradient centrifugation. Anal. Biochem. 159, 122-131 (1986).
  23. Alves, N. J., et al. Functionalized liposome purification via Liposome Extruder Purification (LEP). Analyst. 138, 4746-4751 (2013).
  24. Mayer, L. D., StOnge, G. Determination of free and liposome-associated doxorubicin and vincristine levels in plasma under equilibrium conditions employing ultrafiltration techniques. Anal. Biochem. 232, 149-157 (1995).
  25. Blakeley, B. D., Chapman, A. M., McNaughton, B. R. Split-superpositive GFP reassembly is a fast, efficient, and robust method for detecting protein-protein interactions in vivo. Mol. BioSyst. 8 (2036), (2012).
  26. De Crescenzo, G., Litowski, J. R., Hodges, R. S., O'Connor-McCourt, M. D. Real-time monitoring of the interacation of two-stranded de novo designed coiled-coils: effect of chain length on the kinetic and thermodynamic constants of binding. Biochemistry. 42, 1754-1763 (2003).
  27. Charalambous, A., Antoniades, I., Christodoulou, N., Skourides, P. A. Split-Intein for simultaneous site-specific conjugation of quantum dots to multiple protein targets in vivo. J. Nanobiotechnol. 9, 1-14 (2011).
  28. Lee, E. Y., et al. Global proteomic profiling of native outer membrane vesicles derived fromEscherichia coli. Proteomics. 7, 3143-3153 (2007).
  29. Alves, N. J., Turner, K. B., Medintz, I. L., Walper, S. A. Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles. Sci. Rep. 6, 24866 (2016).
  30. Alves, N. J., Turner, K. B., Medintz, I. L., Walper, S. A. Emerging therapeutic delivery capabilities and challenges utilizing enzyme/protein packaged bacterial vesicles. Ther. Delivery. 6, 873-887 (2015).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved