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Isolation of Inner Membrane Vesicles from Escherichia coli by Using an Affinity Tag
利用亲和标签(Affinity-tag)从大肠杆菌中分离内膜囊泡   

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Abstract

This protocol was developed in a project aimed to identify the inner membrane proteins localizing to cell poles in Escherichia coli (E. coli). By using a known polar protein Tar as a tag, we isolated pole-derived inner membrane vesicles by affinity capture. The specificity of the polar vesicle isolation was confirmed by mass spectrometry that identified more than one hundred proteins, most of which are known inner membrane proteins, including other known polar proteins. This protocol, or if adapted properly by choosing other affinity targets, is well suited to isolate other membrane domains of interest for identification of proteins or lipid composition.

Materials and Reagents

  1. Isopropyl β-D-1-thiogalactopyranoside (IPTG)
  2. Medium copy plasmid pLP8 for recombinant protein expression (Plac lacIq KanR, Li and Young, 2012)
  3. Bacto Tryptone (BD Biosciences, catalog number: 211705 )
  4. Halt protease inhibitor (Thermo Fisher Scientific, catalog number: 78425 )
  5. RNase A (Sigma-Aldrich, catalog number: R5503 )
  6. DNase I (Thermo Fisher Scientific, catalog number: NC9709009 )
  7. anti-FLAG M2 affinity gel (Sigma-Aldrich, catalog number: A2220 )
  8. Poly-Prep chromatography column (Bio-Rad Laboratories, catalog number: 731-1550 )
  9. Micro BCA protein assay kit (Thermo Fisher Scientific, catalog number: 23235 )
  10. Triton X-100
  11. Tween 20
  12. Tryptone medium
  13. Phosphate buffered saline (PBS) (see Recipes)
  14. Phosphate buffered saline with Tween 20 (PBST) (see Recipes)

Equipment

  1. Centrifuges for 1.5 ml, 10 ml and 100 ml volumes
  2. High shear fluid processor (Microfluidics, model: LV1 ) or French press

Procedure

  1. Construct an E. coli strain expressing a tagged recombinant protein. In this experiment, the FLAG-tagged Tar is expressed from an IPTG inducible plasmid.
  2. Transfer 1 ml overnight E. coli culture to 100 ml 1% Tryptone medium and grow at 30 °C. This is the optimal growth condition for flagellar motility and Tar polar localization. Add 25 μM IPTG after 1 h to induce the target protein tagged with a FLAG epitope (Tar-FLAG). Grow the cells for additional 2-3 h.
  3. Pellet the cells (6,000 x g for 5 min at room temperature). In this experiment, low temperature affects the normal polar localization of the Tar protein. Use room temperature in the following steps unless otherwise indicated. Resuspend the pellet in 15 ml PBS. Incubate at 30 °C with shaking for 1 h to allow newly synthesized Tar proteins localizing to poles.
  4. Add 60 μl Halt protease inhibitor. Add 16 μg/ml RNase and 1.3 μg/ml DNase (final concentration). Disrupt the cells with an LV1 high shear fluid processor (Microfluidics) at 20,000 psi (the disruption takes only one sec). Cool the samples immediately on ice.
  5. Spin (10,000 x g for 5 min at 4 °C) to remove unbroken cells.
  6. Transfer 10 ml supernatant containing the membrane vesicles to a 15 ml tube. Add 2 ml PBST and mix by inverting the tube several times.
  7. Add 150 μl anti-FLAG gel beads followed by incubation on ice for 1 h with slow shaking.
  8. Transfer the mixture to an empty Poly-Prep chromatography column. Snap off the seal at the bottom of the column to allow the liquid to flow.
  9. Rinse the beads twice with 8 ml cold PBST.
  10. Cap the bottom of the column. Add 1 ml cold PBST to resuspend the beads. Transfer the mixture to a 1.5 ml tube (this step is important because proteins and vesicles are trapped non-specifically in the bed support layer at the bottom of the column).
  11. Spin (6,000 x g for 1 min at 4 °C) to pellet the beads. Wash twice with 1 ml cold PBST.
  12. Resuspend the beads in 85 μl 1% Triton X-100 in PBS and incubated at room temperature for 10 min to solubilize the inner membranes.
  13. Centrifuge the mixture (6,000 x g for 1 min at room temperature). Transfer the supernatant containing the released proteins to a clean tube for further analysis.
  14. Wash the pellet with 1 ml 1% Triton X-100 in PBS. Resuspend the pellet in 85 μl 1% SDS in water and incubate at 90 °C for 5 min to elute all other proteins, including the FLAG tagged protein. Centrifuge and collect the supernatant (6,000 x g for 1 min at room temperature).
  15. Determine protein concentration in the sample using a Micro BCA protein assay kit.

Recipes

  1. PBS
    40 mM sodium phosphate
    150 mM sodium chloride (pH 7.4)
  2. PBST
    PBS
    0.05% Tween 20

Acknowledgments

Funding for this project was provided by the US Government and managed by the Army Research Office under Award No. W911NF-10-1-0058.

References

  1. Li, G. and Young, K. D. (2012). Isolation and identification of new inner membrane-associated proteins that localize to cell poles in Escherichia coli. Mol Microbiol 84(2): 276-295.
    (This paper was reviewed by Moselio Schaechter in the ASM blog Small Things Considered: Polar Enchantment, May 7, 2012)

简介

该方案是在旨在鉴定定位于大肠杆菌(E.coli)中的细胞极的内膜蛋白的项目中开发的。 通过使用已知的极性蛋白质Tar作为标签,我们通过亲和捕获分离杆来源的内膜囊泡。 极性囊泡分离的特异性通过质谱法证实,其鉴定了超过一百个蛋白质,其中大多数是已知的内膜蛋白,包括其他已知的极性蛋白质。 该方案或者如果通过选择其它亲和靶适当适当地适合于分离其它感兴趣的膜结构域以鉴定蛋白质或脂质组成。

材料和试剂

  1. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)
  2. 用于重组蛋白表达的中等拷贝质粒pLP8(Plac lacIqKanR,Li和Young,2012)
  3. Bacto胰蛋白胨(BD Biosciences,目录号:211705)
  4. Halt蛋白酶抑制剂(Thermo Fisher Scientific,目录号:78425)
  5. RNA酶A(Sigma-Aldrich,目录号:R5503)
  6. DNase I(Thermo Fisher Scientific,目录号:NC9709009)
  7. 抗FLAG M2亲和凝胶(Sigma-Aldrich,目录号:A2220)
  8. Poly-Prep色谱柱(Bio-Rad Laboratories,目录号:731-1550)
  9. Micro BCA蛋白测定试剂盒(Thermo Fisher Scientific,目录号:23235)
  10. Triton X-100
  11. 吐温20
  12. 胰蛋白胨培养基
  13. 磷酸盐缓冲盐水(PBS)(见Recipes)
  14. 含Tween 20的磷酸盐缓冲液(PBST)(见配方)

设备

  1. 离心机为1.5 ml,10 ml和100 ml体积
  2. 高剪切流体处理器(Microfluidics,型号:LV1)或法语压力机

程序

  1. 构造一个。表达标记的重组蛋白的大肠杆菌菌株。在该实验中,FLAG标记的焦油由IPTG诱导型质粒表达
  2. 转移1ml过夜。大肠杆菌培养物至100ml 1%胰蛋白胨培养基并在30℃下生长。这是鞭毛运动和Tar极性定位的最佳生长条件。在1小时后加入25μMIPTG以诱导用FLAG表位(Tar-FLAG)标记的靶蛋白。生长细胞额外2-3小时。
  3. 将细胞沉淀(6,000xg,在室温下5分钟)。在这个实验中,低温影响Tar蛋白的正常极性定位。除非另有说明,否则在以下步骤中使用室温。将沉淀重悬在15ml PBS中。在30℃下振荡孵育1小时,以允许新合成的Tar蛋白定位于杆上
  4. 加入60μlHalt蛋白酶抑制剂。加入16μg/ml核糖核酸酶和1.3μg/ml DNA酶(终浓度)。用LV1高剪切流体处理器(Microfluidics)在20,000psi下破碎细胞(破碎只需一秒钟)。立即在冰上冷却样品。
  5. 在4℃下旋转(10,000xg 5分钟)以除去未破碎的细胞。
  6. 转移10ml含有膜囊泡的上清液到15ml管。 加入2ml PBST,倒置管数次混合
  7. 加入150μl抗FLAG凝胶珠,然后在冰上孵育1小时,缓慢摇动
  8. 将混合物转移至空的Poly-Prep色谱柱。 打开色谱柱底部的密封,让液体流动。
  9. 用8 ml冷PBST冲洗珠子两次。
  10. 盖住色谱柱的底部。 加入1ml冷PBST以重悬珠。 将混合物转移到1.5 ml管中(此步骤很重要,因为蛋白质和囊泡非特异性地捕获在柱底部的床支撑层中)。
  11. 在4℃下旋转(6000xg,1分钟)以使珠粒化。 用1ml冷PBST洗两次。
  12. 将珠子重悬在85μl1%Triton X-100的PBS溶液中,在室温下孵育10分钟,以溶解内膜。
  13. 将混合物离心(6,000xg,在室温下1分钟)。 将含有释放的蛋白质的上清液转移到干净的管中用于进一步分析
  14. 用1ml含1%Triton X-100的PBS洗涤沉淀。 将沉淀重悬于85μl1%SDS的水溶液中,在90°C孵育5分钟,以洗脱所有其他蛋白质,包括FLAG标记的蛋白质。 离心并收集上清液(6000xg,在室温下1分钟)。
  15. 使用Micro BCA蛋白测定试剂盒确定样品中的蛋白浓度

食谱

  1. PBS
    40mM磷酸钠 150mM氯化钠(pH7.4)
  2. PBST
    PBS
    0.05%吐温20

致谢

这个项目的资金由美国政府提供,并由陆军研究办公室管理,奖号W911NF-10-1-0058。

参考文献

  1. Li,G.and Young,K.D。(2012)。 隔离和鉴定新的内膜相关蛋白,其定位于大肠杆菌中的细胞极点 。 Mol Microbiol 84(2):276-295 (本文由Moselio Schaechter在ASM博客中审查小事物:Polar Enchantment,2012年5月7日)
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引用:Li, G. and Young, K. D. (2012). Isolation of Inner Membrane Vesicles from Escherichia coli by Using an Affinity Tag. Bio-protocol 2(20): e273. DOI: 10.21769/BioProtoc.273.
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