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Apr 2013

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Preparation of Outer Membrane Vesicle from Escherichia coli    

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Outer membrane vesicles (OMVs) are spherical bilayered phospholipids of 20-200 nm in size produced from all Gram-negative bacteria and Gram-positive bacteria investigated to date. Previous biochemical and proteomic studies have revealed that the Gram-negative bacteria-derived OMVs are composed of various components like outer membrane proteins, lipopolysaccharides, outer membrane lipids, periplasmic proteins, DNA, and RNA. Here, in this protocol, we describe the method to isolate the OMVs from the culture supernatant of Escherichia coli (E. coli).

Keywords: Outer membrane vesicles, Bacterial extracellular vesicles, Vesicle isolation

Materials and Reagents

  1. Phosphate buffered saline (PBS) (Gibco®, catalog number: 70013-032 )
  2. E. coli (Isolated from the peritoneal lavage fluid of cecal ligation and puncture-operated mice)
  3. Luria-Bertani broth (LB) medium (Merck KGaA, catalog number: 1.10285.0500 ) (see Recipes)


  1. 2 L Flasks
  2. Shaking incubator
  3. Centrifuge
  4. 500 ml Bottle top filter 43 mm neck (0.45 μm and 0.22 μm) (Corning, catalog number: 430514 , 430513 )
  5. QuixStand Benchtop System (Amersham Biosciences, catalog number: 56-4107-44 )
  6. 100-kDa hollow-fiber membrane (Amersham Biosciences, catalog number: 56-4101-33 )
  7. Vacuum pump
    Note: All centrifuge tubes and flasks should be autoclaved before use to avoid contamination


  1. A single colony of E. coli is transferred to 5 ml of LB broth.
  2. The bacteria are incubated in an orbital bacteria shaking incubator at 200 rpm at 37 °C overnight (8 h).
  3. LB broth of 500 ml is inoculated with 1/100 volume of the overnight cultured cells.
    1. Use 2 L flask when culturing 500 ml. Also, since 1/100 volume of 5 ml is 50 μl, before inoculation, increase the volume by adding about 900 μl of fresh LB medium to reduce cell loss.
    2. The yield of OMVs in terms of protein amount is 100 μg per 1 liter of E. coli culture.
  4. The cells are grown for 12 h at 200 rpm at 37 °C.
  5. The cells are pelleted at 5,000 x g for 15 min.
  6. The supernatant fraction is collected and pelleted again at 5,000 x g for 15 min.
  7. The supernatant is collected and filtered through a bottle top filter of pore size 0.45 μm using a vacuum pump.
  8. The filtered supernatant is concentrated to 50-fold by ultra-filtration with a Quixstand Benchtop System using a 100 kDa hollow-fiber membrane.
    Note: Because the yield of OMVs is very low, in order to obtain a visible pellet after ultracentrifugation, the total volume of bacteria culture should be more than 5-7 liters. However, depending on the amount of OMV needed, the volume of bacteria culture could be reduced as well as the degree of concentration.  Ex. 7 L of bacteria culture supernatant is concentrated to give about 280 ml of the concentrated supernatant to be filled in the total of four ultra-centrifuge tube (70 ml each). 
  9. The concentrated supernatant is filtered once again through a 0.22 μm vacuum filter to remove any remaining debris or bacteria.
  10. The resulting filtrate is subjected to ultra-centrifugation at 150,000 x g for 3 h at 4 °C.
  11. The supernatant is removed and the pellet (purified OMV) is resuspended in PBS and stored at – 80 °C until use.

    Figure 1. TEM image of E. coli OMV


  1. LB medium
    1% Tryptone, 0.5% yeast extract, 200 mM NaCl


This protocol was adapted from previously published work (Kim et al., 2013). This work was supported by a grant from the Korean Ministry of Education, Science and Technology, FPR08B1-240 of the 21C Frontier Functional Proteomics Program and Mid-career Researcher Program of National Research Foundation of Korea (NRF) grant funded by the Korea government MEST (No. 20110000215 and No. 20120005634).


  1. Kim, O. Y., Hong, B. S., Park, K. S., Yoon, Y. J., Choi, S. J., Lee, W. H., Roh, T. Y., Lotvall, J., Kim, Y. K. and Gho, Y. S. (2013). Immunization with Escherichia coli outer membrane vesicles protects bacteria-induced lethality via Th1 and Th17 cell responses. J Immunol 190(8): 4092-4102.
  2. Lee, E. Y., Choi, D. S., Kim, K. P. and Gho, Y. S. (2008). Proteomics in gram-negative bacterial outer membrane vesicles. Mass Spectrom Rev 27(6): 535-555.
  3. Park, K. S., Choi, K. H., Kim, Y. S., Hong, B. S., Kim, O. Y., Kim, J. H., Yoon, C. M., Koh, G. Y., Kim, Y. K. and Gho, Y. S. (2010). Outer membrane vesicles derived from Escherichia coli induce systemic inflammatory response syndrome. PLoS One 5(6): e11334.
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Kim, O. Y., Hong, B. S., Park, K., Yoon, Y. J., Choi, S. J., Lee, W. H., Roh, T., Kim, Y. and Gho, Y. S. (2013). Preparation of Outer Membrane Vesicle from Escherichia coli. Bio-protocol 3(23): e995. DOI: 10.21769/BioProtoc.995.

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Hello, when OMVs were filtered through a bottle top filter of pore size 0.45 μm, how to keep the bottle top filter sterile? (Is the device a disposable?). Thank you.
5/6/2020 3:32:37 PM Reply
Won Hee Lee
Life Sciences Department, Pohang University of Science and Technology (POSTECH), Republic of Korea

For preparation OMVs, I used disposable bottle top filter [Corning® bottle-top vacuum filters, cellulose acetate membrane, sterile, pore size 0.45 μm, membrane diam. 70 mm, funnel capacity 500 mL, case of 12, CLS430514-12EA]

5/6/2020 4:55:19 PM

Tran Minh Tuan
School of Biological Sciences, Nanyang Technological University, Singapore, Singapore,
How long would the OMVs be stable at -80 °C vs. 4°C? and what is the best way to test for OMV integrity you would recommend (TEM? protein concentration?). Thank you.
8/19/2018 8:06:29 AM Reply
Oh Youn Kim
Life Sciences Department, Pohang University of Science and Technology (POSTECH), Republic of Korea

According to our previous data on the stability of OMVs in different conditions, both -80 and 4 C showed similar stability. But I would recommend -80 C for long storage. To check the OMV integrity, I recommend analyzing the size of OMV and shape using dynamic light scattering or TEM imaging.

9/17/2018 1:06:26 AM

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