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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).

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Preparation of Outer Membrane Vesicle from Escherichia coli

Microbiology > Microbial cell biology > Organelle isolation
Authors: Oh Youn Kim
Oh Youn KimAffiliation: Life Sciences Department, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
Bio-protocol author page: a1053
Bok Sil Hong
Bok Sil HongAffiliation: Life Sciences Department, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
Bio-protocol author page: a1054
Kyong-Su Park
Kyong-Su ParkAffiliation: Life Sciences Department, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
Bio-protocol author page: a1055
Yae Jin Yoon
Yae Jin YoonAffiliation: Life Sciences Department, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
Bio-protocol author page: a1056
Seng Jin Choi
Seng Jin ChoiAffiliation: Life Sciences Department, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
Bio-protocol author page: a1057
Won Hee Lee
Won Hee LeeAffiliation: Life Sciences Department, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
Bio-protocol author page: a1058
Tae-Young Roh
Tae-Young RohAffiliation: Life Sciences Department, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
Bio-protocol author page: a1059
Yoon-Keun Kim
Yoon-Keun KimAffiliation: Life Sciences Department, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
Bio-protocol author page: a1060
 and Yong Song Gho
Yong Song GhoAffiliation: Life Sciences Department, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
For correspondence: ysgho@postech.ac.kr
Bio-protocol author page: a1061
Vol 3, Iss 23, 12/5/2013, 4379 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.995

[Abstract] 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)

Equipment

  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

Procedure

  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.
    Notes:
    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

Recipes

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

Acknowledgments

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).

References

  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.


How to cite this protocol: 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; Full Text



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