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Biotinylation and Purification of Surface-exposed Helicobacter pylori Proteins
表面暴露幽门螺杆菌蛋白的生物素化和纯化   

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Abstract

Interactions between pathogenic bacteria and host cells are often mediated by proteins found on the surfaces of the bacteria. The Gram-negative bacterium Helicobacter pylori is predicted to produce at least 50 surface-exposed outer membrane proteins, but there has been relatively little progress in experimentally analyzing the cell-surface proteome of this organism. Herein, we describe in detail a protocol that allows biotinylation and purification of surface-exposed H. pylori proteins. A comparative analysis of surface-exposed proteins identified by this biotinylation-based approach and by several other independent methods is described in a recent publication (Voss et al., 2014).

Keywords: Outer membrane proteins(外层膜蛋白), Autotransporter(汽车运输船), Type V secretion(V型分泌), Adhesin(最高), Helicobacter pylori(幽门螺杆菌)

Materials and Reagents

  1. Tryptone (BD, catalog number: 211705 )
  2. Proteose peptone #3 (BD, catalog number: 211693 )
  3. Yeast Extract (BD, catalog number: 212720 )
  4. NaCl (Acros Organics, catalog number: 207790250 )
  5. Dextrose (Thermo Fisher Scientific, catalog number: D14-212 )
  6. Fetal bovine serum (Atlanta Biologicals, catalog number: S11150 )
  7. Tris Base (Thermo Fisher Scientific, catalog number: BP152 )
  8. Na2HPO4 (Sigma-Aldrich, catalog number: S5136 )
  9. KCl (Sigma-Aldrich, catalog number: P-9333 )
  10. 10x PBS (Corning, catalog number: 46-013-CM )
  11. MgCl2.6H2O (Thermo Fisher Scientific, catalog number: M35 )
  12. CaCl2 (Sigma-Aldrich, catalog number: C-4901 )
  13. EDTA-Na2.2H2O (Sigma-Aldrich, catalog number: E4884 )
  14. D-biotin (Sigma-Aldrich, catalog number: B4501 )
  15. S-NHS-LC-Biotin (Thermo Fisher Scientific, catalog number: 21335 )
  16. Zwittergent 3-14 (Fluka, catalog number: 40772 )
  17. Protease inhibitor cocktail (Roche Diagnostics, catalog number: 04693159001 )
  18. Monomeric avidin magnetic beads, Blocking buffer, Regeneration buffer (Bioclone, catalog number: MMI-101 )
  19. 100% (w/v) TCA (Sigma-Aldrich, catalog number: T0699 )
  20. Acetone (Thermo Fisher Scientific, catalog number: A16F )
  21. Brucella broth (see Recipes)
  22. Biotinylation buffer (see Recipes)
  23. TNKCM (see Recipes)
  24. Lysis buffer (see Recipes)
  25. TKE (see Recipes)
  26. TKEZ (see Recipes)
  27. Sample dilution buffer (see Recipes)
  28. Blocking buffer (see Recipes)
  29. Regeneration buffer (see Recipes)
  30. Wash buffer (see Recipes)
  31. Elution buffer (see Recipes)

Equipment

  1. CO2 shaking incubator (ATR Biotech, model: AJ125B )
  2. Bench top centrifuge (Thermo Fisher Scientific, catalog number: 75004381 )
  3. Sonicator (Thermo Fisher Scientific, catalog number: FB505 )
  4. Bench top centrifuge (Eppendorf, catalog number: 5424 )
  5. Ultracentrifuge (Beckman Coulter, catalog number: A94469 )
  6. Barnstead/Thermolyne Labquake Shaker Rotisserie (Labquake, catalog number: C400110 )
  7. DynaMagTM- Spin Magnet (Life Technologies, catalog number: 12320D )

Procedure

  1. On-cell biotinylation
    1. Grow 25 ml of H. pylori liquid culture in Brucella broth containing 10% fetal bovine serum until late-log phase (OD600 = 0.7) at 37 °C.
    2. Pellet all bacteria from the 25 ml culture at 3,500 x g for 20 min at 4 °C, and discard media.
    3. Resuspend bacterial pellet in 10 ml Biotinylation buffer.
    4. Pellet bacteria at 3,500 x g for 10 min at 4 °C, and discard buffer.
    5. Resuspend bacterial pellet in 10 ml Biotinylation buffer.
    6. Add S-NHS-LC-biotin to a final concentration of 200 μM.
    7. Incubate on ice for 30 min.
    8. Quench reaction by adding 20 ml TNKCM, incubate at room temperature for 10 min.
    9. Pellet bacteria at 3,500 x g for 20 min at 4 °C, and discard buffer.
    10. Resuspend bacterial pellet in 10 ml TNKCM, pellet bacteria at 3,500 x g for 10 min at 4 °C, and discard buffer. Repeat two more times for a total of three washes.

  2. Bacterial lysis and subcellular fractionation
    1. Resuspend bacterial pellet in 3 ml lysis buffer + protease inhibitor cocktail.
    2. Sonicate (4 times, 10 sec each, at 25% maximum amplitude) on ice to lyse bacteria, pellet intact bacteria at 7,000 x g for 10 min, collect lysis supernatant.
    3. Pellet membranes by spinning lysate at 40,000 x g for 30 min at 4 °C.
    4. Discard supernatant.
    5. Rinse membrane pellet with TKE three times.
    6. Collect membrane pellet by scraping with a clean pipette tip and resuspend in 1 ml TKEZ + protease inhibitor cocktail.
    7. Solubilize for 1 h at 4 °C with head-over-head mixing.
    8. Spin at 100,000 x g for 1 h at 4 °C, collect supernatant.

  3. Isolation and enrichment of biotinylated proteins
    1. Dilute the supernatant 10x with sample dilution buffer (resulting in a volume of 10 ml).
    2. Aliquot 50 μl of avidin magnetic bead suspension into a microfuge tube.
    3. Place tube on magnet, remove storage buffer.
    4. Resuspend beads in 500 μl PBS, place on magnet and discard buffer.
    5. Resuspend beads in 200 μl blocking buffer.
    6. Incubate beads for 10 min with head-over-head mixing.
    7. Place on magnet, discard buffer, resuspend beads in 400 μl regeneration buffer.
    8. Place on magnet, discard buffer, resuspend beads in 500 μl wash buffer.
    9. Place on magnet, discard buffer.
    10. Add 1 ml aliquot of sample described in step C19 to equilibrated beads and incubate at room temperature with head over head mixing for 10 min. Then place the tube on magnet, discard supernatant, and apply another 1 ml aliquot. Repeat this procedure until the entire 10 ml of sample is applied to the beads.
    11. After entire 10 ml sample from step C19 has been incubated with the magnetic beads, resuspend beads in 1 ml wash buffer, and incubate for 5 min with head-over-head mixing. Then place the tube on magnet and discard supernatant. Repeat two more times for a total of three washes.
    12. Resuspend beads in 1 ml wash buffer and transfer the suspended beads to a clean tube.
    13. To elute biotinylated proteins: place the bead suspension on magnet, discard supernatant, resuspend beads in 1 ml elution buffer, and incubate at room temperature for 10 min with head-over-head mixing.
    14. Place the tube on magnet and save the supernatant. Add another 1 ml elution buffer to the beads and incubate at room temperature for 10 min with head over head mixing. Then place on the magnet and again save the supernatant. Repeat this procedure once more, resulting in a total elution volume of 3 ml.
    15. Precipitate proteins by adding 1 ml TCA to the 3 ml elution (25% final concentration TCA).
    16. Vortex to mix, incubate at 4 °C for at least 30 min. (Overnight storage at 4 °C is possible at this stage).
    17. Spin at 21,000 x g for 30 min at 4 °C, discard supernatant.
    18. Add 1 ml ice cold acetone, spin at 21,000 x g at 4 °C for 10 min, discard supernatant, repeat once for a total of two acetone washes.
    19. Air dry pellet at room temperature, store at -20 °C until needed.

Notes

  1. Samples can be initially analyzed by SDS-PAGE and silver staining, or by Western blotting if suitable antisera reactive with H. pylori outer membrane proteins are available. A comprehensive identification of the proteins present in preparations of purified biotinylated proteins can be accomplished by mass spectrometry. Results obtained using this method for biotinylation and purification of surface-exposed H. pylori proteins, along with a comparative analysis of results obtained using other methods, are described in detail in Voss et al. (2014). The use of multiple methods for identifying surface-exposed proteins helps to minimize false-positive results.
  2. In parallel with the purification of biotinylated proteins from biotinylated bacteria, we recommend that non-biotinylated bacteria should be processed as a control as described previously in Voss et al. (2014). This comparative approach minimizes false-positive results that can arise due to non-specific protein binding to beads.

Recipes

  1. Brucella broth
    10 g/L tryptone
    10 g/L proteose peptone #3
    5 g/L NaCl
    2 g/L yeast extract
    1 g/L dextrose
  2. Biotinylation buffer
    1x PBS
    1 mM CaCl2
    0.5 mM MgCl2
    1.6 mM D-biotin
    pH 7.4
  3. TNKCM (for quenching of S-NHS-LC-biotin and washing bacteria)
    50 mM Tris (pH 7.4)
    100 mM NaCl
    27 mM KCl
    1 mM CaCl2
    0.5 mM MgCl2
  4. Lysis buffer
    50 mM Tris (pH 7.4)
    1 mM MgCl2
  5. TKE (for rinsing membrane pellet)
    50 mM Tris (pH 7.4)
    150 mM KCl
    10 mM EDTA
  6. TKEZ (for preferentially solubilizing outer membrane proteins)
    50 mM Tris (pH 7.4)
    150 mM KCl
    10 mM EDTA
    2% Zwittergent 3-14
  7. Sample dilution buffer
    100 mM Na2HPO4 (pH 7.4)
    150 mM NaCl
  8. Blocking buffer
    100 mM Na2HPO4 (pH 7)
    150 mM NaCl
    2 mM D-biotin
  9. Regeneration buffer
    100 mM glycine (pH 2.8)
  10. Wash buffer (for washing magnetic beads)
    100 mM Na2HPO4 (pH 7.4)
    150 mM NaCl
    0.2% Zwittergent 3-14
  11. Elution buffer
    100 mM Na2HPO4 (pH 7.4)
    150 mM NaCl
    0.2% Zwittergent 3-14
    5 mM D-biotin

Acknowledgments

The biotinylation protocol described here and in Voss et al. (2014) is modified from a protocol described in Sabarth et al. (2002). Supported by the Department of Veterans Affairs, Merit Review grant 2I01 BX000627.

References

  1. Sabarth, N., Lamer, S., Zimny-Arndt, U., Jungblut, P. R., Meyer, T. F. and Bumann, D. (2002). Identification of surface proteins of Helicobacter pylori by selective biotinylation, affinity purification, and two-dimensional gel electrophoresis. J Biol Chem 277(31): 27896-27902.
  2. Voss, B. J., Gaddy, J. A., McDonald, W. H. and Cover, T. L. (2014). Analysis of surface-exposed outer membrane proteins in Helicobacter pylori. J Bacteriol 196(13): 2455-2471.

简介

病原细菌和宿主细胞之间的相互作用通常由在细菌表面上发现的蛋白介导。 革兰氏阴性细菌幽门螺杆菌预计产生至少50个表面暴露的外膜蛋白,但是在实验分析该生物体的细胞表面蛋白质组中相对较少的进展。 在本文中,我们详细描述了允许表面暴露的H的生物素化和纯化的方案。 幽门螺杆菌蛋白。 在最近的出版物(Voss等人,2014)中描述了通过这种基于生物素化的方法和几种其它独立方法鉴定的表面暴露蛋白的比较分析。

关键字:外层膜蛋白, 汽车运输船, V型分泌, 最高, 幽门螺杆菌

材料和试剂

  1. 胰蛋白胨(BD,目录号:211705)
  2. 蛋白胨#3(BD,目录号:211693)
  3. 酵母提取物(BD,目录号:212720)
  4. NaCl(Acros Organics,目录号:207790250)
  5. 右旋糖(Thermo Fisher Scientific,目录号:D14-212)
  6. 胎牛血清(Atlanta Biologicals,目录号:S11150)
  7. Tris碱(Thermo Fisher Scientific,目录号:BP152)
  8. Na 2 HPO 4(Sigma-Aldrich,目录号:S5136)
  9. KCl(Sigma-Aldrich,目录号:P-9333)
  10. 10x PBS(Corning,目录号:46-013-CM)
  11. (Thermo Fisher Scientific,目录号:M35)
  12. CaCl 2(Sigma-Aldrich,目录号:C-4901)
  13. EDTA-Na 2 O 2·2H 2 O(Sigma-Aldrich,目录号:E4884)。
  14. D-生物素(Sigma-Aldrich,目录号:B4501)
  15. S-NHS-LC-生物素(Thermo Fisher Scientific,目录号:21335)
  16. Zwittergent 3-14(Fluka,目录号:40772)
  17. 蛋白酶抑制剂混合物(Roche Diagnostics,目录号:04693159001)
  18. 单体抗生物素蛋白磁珠,封闭缓冲液,再生缓冲液(Bioclone,目录号:MMI-101)
  19. 100%(w/v)TCA(Sigma-Aldrich,目录号:T0699)
  20. 丙酮(Thermo Fisher Scientific,目录号:A16F)
  21. 布鲁氏肉汤(见配方)
  22. 生物素化缓冲液(参见配方)
  23. TNKCM(参见食谱)
  24. 裂解缓冲液(见配方)
  25. TKE(参见食谱)
  26. TKEZ(参见食谱)
  27. 样品稀释缓冲液(参见配方)
  28. 阻止缓冲区(参见配方)
  29. 再生缓冲区(参见配方)
  30. 洗涤缓冲液(见配方)
  31. 洗脱缓冲液(参见配方)

设备

  1. CO 2振荡培养箱(ATR Biotech,型号:AJ125B)
  2. 台式离心机(Thermo Fisher Scientific,目录号:75004381​​)
  3. 超声波仪(Thermo Fisher Scientific,目录号:FB505)
  4. 台式离心机(Eppendorf,目录号:5424)
  5. 超速离心机(Beckman Coulter,目录号:A94469)
  6. Barnstead/Thermolyne Labquake Shaker Rotisserie(Labquake,目录号:C400110)
  7. DynaMag TM - Spin Magnet(Life Technologies,目录号:12320D)

程序

  1. 细胞生物素化
    1. 生长25ml的H。幽门螺杆菌液体培养物在含有10% 胎牛血清,直至晚对数期(OD <600 = 0.7)。
    2. 将来自25ml培养物的所有细菌以3,500×g在4℃下沉淀20分钟,并丢弃培养基。
    3. 将细菌沉淀重悬在10ml生物素化缓冲液中
    4. 在4℃下在3,500×g下沉淀细胞10分钟,并丢弃缓冲液。
    5. 将细菌沉淀重悬在10ml生物素化缓冲液中
    6. 加入S-NHS-LC-生物素至终浓度为200μM
    7. 在冰上孵育30分钟。
    8. 通过加入20ml TNKCM进行淬灭反应,在室温下孵育10分钟
    9. 在4℃下将细胞以3,500×g离心20分钟,并丢弃缓冲液。
    10. 将细菌沉淀重悬在10ml TNKCM中,在4℃下将细菌以3500×g离心10分钟,弃去缓冲液。重复两次以上的a  共三次洗涤。

  2. 细菌裂解和亚细胞分馏
    1. 在3ml裂解缓冲液+蛋白酶抑制剂混合物中重悬细菌沉淀
    2. 在冰上超声(4次,每次10秒,25%最大振幅)  裂解细菌,以7,000×g离心10分钟沉淀完整细菌,收集 裂解上清液
    3. 通过在4℃下以40,000×g离心裂解物30分钟来沉淀膜。
    4. 弃去上清液。
    5. 用TKE冲洗膜沉淀三次。
    6. 通过用干净的移液管尖端刮擦收集膜沉淀,并重悬于1ml TKEZ +蛋白酶抑制剂混合物中。
    7. 在4℃下溶解1小时
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Voss, B. J. and Cover, T. L. (2015). Biotinylation and Purification of Surface-exposed Helicobacter pylori Proteins. Bio-protocol 5(8): e1455. DOI: 10.21769/BioProtoc.1455.
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