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Preparation of Outer Membrane Vesicle from Escherichia coli
从大肠杆菌分离外膜囊泡   

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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(外膜囊泡), Bacterial extracellular vesicles(细菌胞外囊泡), Vesicle isolation(囊泡分离)

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.

简介

外膜囊泡(OMV)是从所有革兰氏阴性细菌和革兰氏阳性细菌产生的20-200nm的球形双层磷脂,迄今为止研究。 以前的生物化学和蛋白质组学研究已经显示,革兰氏阴性细菌衍生的OMV由各种组分组成,如外膜蛋白,脂多糖,外膜脂质,周质蛋白,DNA和RNA。 在这里,在该方案中,我们描述了从大肠杆菌的培养物上清液中分离OMV的方法(大肠杆菌)。

关键字:外膜囊泡, 细菌胞外囊泡, 囊泡分离

材料和试剂

  1. 磷酸盐缓冲盐水(PBS)(Gibco ,目录号:70013-032)
  2. E。 大肠杆菌(从盲肠结扎和穿刺操作的小鼠的腹膜灌洗液中分离)
  3. Luria-Bertani肉汤(LB)培养基(Merck KGaA,目录号:1.10285.0500)(参见配方)

设备

  1. 2升烧瓶
  2. 摇动培养箱
  3. 离心机
  4. 500ml瓶顶过滤器43mm颈(0.45μm和0.22μm)(Corning,目录号:430514,430513)
  5. QuixStand Benchtop System(Amersham Biosciences,目录号:56-4107-44)
  6. 100-kDa中空纤维膜(Amersham Biosciences,目录号:56-4101-33)
  7. 真空泵
    注意:所有离心管和烧瓶在使用前应进行高压灭菌,以避免污染。

程序

  1. 将大肠杆菌的单个菌落转移到5ml LB肉汤中。
  2. 将细菌在轨道细菌摇动培养箱中在37℃下以200rpm温育过夜(8小时)
  3. 将500ml LB肉汤用1/100体积的过夜培养的细胞接种 注意:
    1. 培养500ml时使用2L烧瓶。 另外,由于5ml的1/100体积为50μl,因此在接种前,通过加入约900μl新鲜LB培养基来增加体积以减少细胞损失。
    2. 根据蛋白质量,OMV的产量为每1升大肠杆菌培养物100μg。
  4. 细胞在37℃下以200rpm生长12小时。
  5. 将细胞在5,000xg下沉淀15分钟。
  6. 收集上清液级分并再次在5,000xg下沉淀15分钟。
  7. 收集上清液并使用真空泵通过孔径0.45μm的瓶顶过滤器过滤。
  8. 通过使用100kDa中空纤维膜的Quixstand Benchtop系统进行超滤,将过滤的上清液浓缩至50倍。
    注意:由于OMV的产量非常低,为了在超速离心后获得可见的沉淀,细菌培养物的总体积应当大于5-7升。然而,根据所需的OMV的量,可以减少细菌培养物的体积以及浓度的程度。例如,将7L细菌培养物上清液浓缩,得到约280ml浓缩的上清液,将其装入总共四个超离心管(每个70ml)中。</em>
  9. 将浓缩的上清液再次通过0.22μm真空过滤器过滤以除去任何残留的碎片或细菌。
  10. 将所得滤液在4℃下以150,000×g超速离心3小时。
  11. 除去上清液,将沉淀(纯化的OMV)重悬于PBS中,并储存在-80℃直至使用。


    图1.E的TEM图像。大肠杆菌 OMV

食谱

  1. LB培养基
    1%胰蛋白胨,0.5%酵母提取物,200mM NaCl

致谢

此协议改编自以前发表的作品(Kim等人,2013年)。 这项工作得到了韩国教育,科学技术部的资助,21C前沿功能蛋白质组学项目的FPR08B1-240和韩国国家研究基金会(NRF)的中期职业生涯研究员资助由韩国政府MEST资助 (No.20110000215和No.20120005634)。

参考文献

  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, 用大肠杆菌外膜囊免疫可通过Th1保护细菌诱导的致死性和Th17细胞应答。 J Immunol 190(8):4092-4102。
  2. Lee,E.Y.,Choi,D.S.,Kim,K.P。和Gho,Y.S。(2008)。 革兰氏阴性细菌外膜囊泡中的蛋白质组学。 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, 来自大肠杆菌的外膜囊泡诱导全身性炎症反应综合征。/a> PLoS One 5(6):e11334。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: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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