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Preparation of Everted Membrane Vesicles from Escherichia coli Cells
利用大肠埃希杆菌细胞制备反转膜囊泡   

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Abstract

The protocol for obtaining electrically sealed membrane vesicles from E. coli cells is presented. Proton pumps such as Complex I, quinol oxidase, and ATPase are active in the obtained vesicles. Quality of the preparation was tested by monitoring the electric potential generated by these pumps.

Keywords: Escherichia coli(大肠埃希杆菌), Membrane vesicles(膜囊泡), Proton pumping(质子泵)

Background

Studying of membrane enzymes often requires embedding them into the natural lipid environment. Inside-out, everted membrane vesicles allowed to explore effects of different substrates, inhibitors and other ligands on the operation of these enzymes. Functioning proton pumps, such as NADH: ubiquinone oxidoreductase Type 1 (Complex I), quinol oxidase, and ATPase also requires good electrical sealing of the vesicles. This protocol provides sufficient results in preparation of such vesicles for studying these enzymes (e.g., Euro et al., 2008; Belevich et al., 2011).

Materials and Reagents

  1. Glass test tubes (approximately 10 x 90 mm)
  2. E. coli MWC215 (SmR ndh::CmR) or GR70N (wild type or mutated) cells grown under aeration
  3. Luria Broth or other medium suitable for aerobic growth of E. coli culture
  4. Antibiotics, as required for particular E. coli strain
  5. Lysozyme
  6. Ethylenediaminetetraacetic acid (EDTA)
  7. Magnesium sulfate (MgSO4)
  8. Dithiothreitol (DTT)
  9. Phenylmethylsulfonyl fluoride (PMSF)
  10. Ethanol
  11. Liquid N2
  12. Electric potential-sensitive probe Oxonol VI (Sigma-Aldrich, catalog number: 75926 )
  13. Ammonium sulfate, (NH4)2SO4
  14. Decylubiquinone (Sigma-Aldrich, catalog number: D7911 )
  15. Potassium cyanide (KCN)
  16. Nicotinamide adenine dinucleotide reduced (NADH)
  17. Rolliniastatin
  18. Ubiquinone 1 (Sigma-Aldrich, catalog number: C7956 )
  19. Adenosine triphosphate (ATP)
  20. Aurovertin B (Sigma-Aldrich, catalog number: A5297 )
    Note: This product has been discontinued.
  21. Tris-HCl
  22. Sucrose
  23. HEPES
  24. Potassium hydroxide (KOH)
  25. Potassium chloride (KCl)
  26. Magnesium chloride (MgCl2)
  27. 3-(N-morpholino)propanesulfonic acid (MOPS)
  28. 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP)
  29. The medium (see Recipes)
  30. Buffer A (see Recipes)
  31. Buffer B (see Recipes)
  32. Buffer C (see Recipes)
  33. Buffer D (see Recipes)

Equipment

  1. 200 ml Erlenmeyer flasks
  2. 2 L Erlenmeyer flasks
  3. Shaker, such as Sertomat R/Sertomat HR or other suitable
  4. Centrifuges, such as (Thermo Fisher Scientific, model: Sorvall RC 5C Plus )
  5. Rotors (Thermo Fisher Scientific, Thermo ScientificTM, models: SS-34 , SLA 3000 ; Beckman Coulter, model: Ti70 )
  6. Sonicator (Emerson, model: Branson Sonifier Cell Disruptor B15 ) or other with the tip diameter 10-12 mm
  7. UV/VIS spectrophotometer (i.e., Ocean Optics, model: HR2000 )

Procedure

  1. Cells
    1. Inoculate the cells into 5-100 ml (dependently on how much you want to grow next day) LB (+ antibiotics dependently on the strain) to get overnight culture (RT, orbital shaker).
    2. Inoculate overnight culture 1:50 to LB (with antibiotics if necessary) in 50 ml to 200 ml Erlenmeyer flasks or 700 ml LB in 2 L Erlenmeyer flasks and grow in orbital shaker at 37 °C, 220-230 rpm for ~4-5 h (A600 ~2.5 for wild type). Follow the growth: cells must be harvested in the late exponential phase. Do not overgrow, otherwise the cell wall would be thicker and there could be problem with cell disintegration.
    3. Pellet cells at 7,500 x g (8,000 rpm) x 10 min in SS-34 or 8,000 rpm x 20 min in SLA 3000 or other appropriate rotor (10-20 min at approximately 8,000 x g).
  2. Spheroplasts
    1. Suspend cell pellet at RT in 10 ml (2 x 50 ml culture) or 100 ml (3 x 700 ml culture) in buffer A (see Recipe 1).
    2. Add 100 μg/ml lysozyme (10 mg/ml stock solution) and incubate at RT until cells become osmosensitive (don’t treat the cells longer than necessary), approx. 5-10 min. On that purpose perform the osmosensitivity test. Place 50 μl cells to 1 ml 10 mM EDTA, pH 7.0 in the glass tube. Visually seen lysis (the suspension is getting transparent) should occur in 1-2 min. The cells without lysozyme are not disrupted under these conditions.
      Note: Remember to take control before lysozyme addition! The cells without lysozyme stay intact and should be used for comparison with lysozyme-treated cells by matching of the turbidity of the cell suspension.
    3. Add 4 mM MgSO4 (from 1 M stock solution) and traces of DNase. The suspension viscosity should decrease almost immediately.
    4. Pellet spheroplasts at 7,500 x g (8,000 rpm) x 10 min in SS-34.
    5. Suspend the spheroplasts on ice in 20 ml or 40 ml of cold buffer B (see Recipe 2).
    6. Add 2 mM dithiothreitol (DTT) (from 100 mM stock solution), 0.5 mM PMSF (from 100 mM fresh stock solution).
      Notes:
      1. PMSF has short life time in water solutions. It is stable only in absolute ethanol kept anaerobically, therefore the best way is to make fresh stock solution in 95% ethanol.
      2. This medium is optional and can be changed dependently on further experiments, however, high [Mg2+], dithiothreitol (DTT) and protease inhibitor PMSF always significantly improve the membrane quality. Buffer B is developed for measurements of intravesicular acidification by proton pumps. For monitoring the electric potential generation buffer C with low salts concentration was used (see below).
  3. Membranes (on ice/water bath)
    1. Sonicate spheroplasts at approximately 75 W (watts). For efficient sonication the distance between the sonicator tip and walls of the tube with spheroplasts suspension should not be wider than 6-8 mm. To prevent overheating 50% sonication pulses are used. After approx. 30 sec sonication it is necessary to make 1 min break for cooling. For osmosensitive cells this should be repeated 4-6 times. The cell disruption can be visually controlled.
    2. Pellet undisrupted cells and cell debris at 7,200 x g x 10 min in SS-34 and discard it.
    3. Pellet membranes in Ti70 at 150,000 x g (40,000 rpm) x 40 min or other appropriate rotor at 80,000-150,000 x g for 30-40 min. If the membrane vesicles with larger internal volume are required the sedimentation should proceed shorter or at lower RCF (relative centrifugal force).
    4. Rinse the pellet if necessary and suspend membranes (in minimal volume) of desired buffer B (see Recipe 2).
    5. If the membranes are not used immediately freeze them in aliquots in liquid N2 and store at -80 °C.
    6. The quality of obtained membrane vesicles can be simply tested by monitoring the electric potential (∆ψ) generated by proton pumps using ∆ψ-sensitive probe, Oxonol VI, as shown in Figure1. The value of ∆ψ can be estimated by calibration of absorption changes with imposed diffusion potentials (i.e., Ghelli et al., 1997; Pouliquin et al., 1999). H+ or K+ diffusion potential could be established in the presence of proton uncoupler such as carbonyl cyanide m-chlorophenyl hydrazine (CCCP) or potassium transporter valinomycin, correspondingly. The value of the diffusion potential can be calculated by the Nernst equation. The detailed description of the procedure can be found in the paper by Ghelli et al., 1997.
    7. The NADH:ubiquinone reductase activity of membrane-bound Complex I can be measured in buffer C at 30 °C by following NADH oxidation at 340 nm (ε = 6.2 mM-1 cm-1). The NADH:ubiquinone oxidoreductase activity of Complex I in the membrane vesicles prepared from E. coli GR70N is 0.92 ± 0.13 (n = 10) (Belevich et al., 2011).


      Figure 1. Following the electric potential generation by three proton pumps in E. coli membranes using ∆ψ-sensitive probe Oxonol VI. The membrane vesicles were loaded with buffer D. The medium consists of buffer C accomplished with 5 mM (NH4)2SO4 (to convert ∆pH to ∆ψ) and Oxonol VI 2.5 μM (stored in 2-5 mM ethanol or methanol stock solution at -20 °C, stable at least a half of year). The membranes were added at concentration 80 μg/ml. The reaction was followed by the measurements of absorption changes as indicated. To monitor ∆ψ generation by NADH:ubiquinone oxidoreductase, Type I, (Complex I), the medium was accomplished with 100 μM decylubiquinone and 1 mM KCN to block the terminal oxidase. 50 μM NADH was added to start the reaction, ∆ψ was dissipated by Complex I inhibitor, rolliniastatin (Roll) at concentration 1 μM. To monitor ∆ψ generation by quinol oxidase bo3 the medium was accomplished with 50 μM ubiquinone 1. To start the reaction 2 mM dithiothreitol was added, ∆ψ was dissipated by 1 mM KCN addition. To monitor ∆ψ generation by ATPase the medium was accomplished with 1 mM MgSO4. The reaction was initiated by an addition of 1 mM ATP, ∆ψ was dissipated by an addition of ATPase inhibitor, aurovertin B (Auro B) at concentration 10 μM.

Notes

  1. This protocol gives highly reproducible results, however, all procedures should be conducted as fast as possible. Breaks (especially after the cells disruption step) may cause an increase in proton leakage through the membrane and decrease in membrane enzymes activity.
  2. This protocol was developed especially for E. coli cells and it should not be used for other bacterial species without modifications.

Recipes

  1. Buffer A
    200 mM Tris-HCl, pH 8.0
    2 mM EDTA
    30% sucrose
  2. Buffer B
    100 mM HEPES-KOH, pH 7.5
    100 mM KCl
    10 mM MgCl2
  3. Buffer C
    25 mM HEPES-BTP, pH 7.5
    3 mM KCl
  4. Buffer D
    100 mM MOPS-BTP, pH 7.0
    1 mM MgSO4

Acknowledgments

This work was supported by grants from Biocentrum Helsinki, the Sigrid Juselius Foundation, and the Academy of Finland.

References

  1. Euro, L., Belevich, G., Verkhovsky, M. I., Wikström, M. and Verkhovskaya. M. (2008). Conserved lysine residues of the membrane subunit NuoM are involved in energy conversion by the proton-pumping NADH:ubiquinone oxidoreductase (Complex I). Biochim Biophys Acta 1777(9): 1166-1172.
  2. Belevich, G., Knuuti, J., Verkhovsky, M.I., Wikström, M, Verkhovskaya, M. (2011). Probing the mechanistic role of the long α-helix in subunit L of respiratory Complex I from Escherichia coli by site-directed mutagenesis. Mol Microbiol 82(5): 1086-1095.
  3. Ghelli, A., Benelli, B. and Esposti, M. D. (1997). Measurement of the membrane potential generated by complex I in submitochondrial particles. J Biochem 121(4): 746-755.
  4. Pouliquin, P., Grouzis, J. and Gibrat, R. (1999). Electrophysiological study with oxonol VI of passive NO3- transport by isolated plant root plasma membrane. Biophys J 76: 360-373.

简介

用于从E获得电密封的膜囊泡的方案。 呈现大肠杆菌细胞。 质子泵如复合物I,喹啉氧化酶和ATP酶在获得的囊泡中是活性的。 通过监测这些泵产生的电位来测试制备的质量。

研究膜酶通常需要将它们嵌入天然脂质环境中。 内向外的旋转膜囊泡可以探索不同底物,抑制剂和其他配体对这些酶的操作的影响。 功能性的质子泵,如NADH:泛醌氧化还原酶1型(复合物I),喹啉氧化酶和ATP酶也需要良好的电气密封囊泡。 该方案在制备用于研究这些酶的这种囊泡方面提供了足够的结果(例如,欧洲等人,2008; Belevich等人,2011)。

关键字:大肠埃希杆菌, 膜囊泡, 质子泵

材料和试剂

  1. 玻璃试管(约10 x 90 mm)
  2. 电子。大肠杆菌在通气下生长的MWC215(Sm R ndh :: Cm R )或GR70N(野生型或突变型)细胞
  3. Luria肉汤或适用于E的有氧生长的其他培养基。大肠杆菌文化
  4. 抗生素,特别是E。大肠杆菌菌株
  5. 溶菌酶
  6. 乙二胺四乙酸(EDTA)
  7. 硫酸镁(MgSO 4)
  8. 二硫苏糖醇(DTT)
  9. 苯甲磺酰氟(PMSF)
  10. 乙醇
  11. 液体N 2
  12. 电势敏感探针Oxonol VI(Sigma-Aldrich,目录号:75926)
  13. 硫酸铵(NH 4)2 SO 4
  14. Decylubiquinone(Sigma-Aldrich,目录号:D7911)
  15. 氰化钾(KCN)
  16. 烟酰胺腺嘌呤二核苷酸减少(NADH)
  17. 罗利尼他汀
  18. 泛醌1(Sigma-Aldrich,目录号:C7956)
  19. 三磷酸腺苷(ATP)
  20. Aurovertin B(Sigma-Aldrich,目录号:A5297)
    注意:本产品已停产。
  21. Tris-HCl
  22. 蔗糖
  23. HEPES
  24. 氢氧化钾(KOH)
  25. 氯化钾(KCl)
  26. 氯化镁(MgCl 2)
  27. 3-(N-吗啉代)丙磺酸(MOPS)
  28. 1,3-双(三(羟甲基)甲基氨基)丙烷(BTP)
  29. 介质(见配方)
  30. 缓冲液A(参见食谱)
  31. 缓冲液B(参见食谱)
  32. 缓冲区C(见配方)
  33. 缓冲区D(见配方)

设备

  1. 200毫升锥形瓶
  2. 2升三角烧瓶
  3. 振荡器,如Sertomat R/Sertomat HR或其他合适的
  4. 离心机,如(赛默飞世尔科技,型号:Sorvall RC 5C Plus)
  5. 转子(Thermo Fisher Scientific,Thermo Scientific TM,型号:SS-34,SLA 3000; Beckman Coulter,型号:Ti70)
  6. 超声波发生器(艾默生,型号:Branson Sonifier Cell Disruptor B15)或其他尖端直径10-12 mm
  7. UV/VIS分光光度计(,Ocean Optics,型号:HR2000)

程序

  1. 细胞
    1. 将细胞接种到5-100毫升(取决于你第二天要生长多少)LB(+抗生素依赖于菌株)获得过夜培养(RT,轨道摇床)。
    2. 在50ml至200ml锥形瓶中的LB(用抗生素(如果需要))接种过夜培养物1:50,并在37℃,220-230rpm下在轨道摇床中生长〜4-5 h(野生型的A <600>〜2.5)。遵循增长:细胞必须在晚期指数期收获。不要过度生长,否则细胞壁会变厚,细胞分解就会有问题。
    3. 在SLA 3000或其他合适的转子中以7,000 xg(8,000rpm)×10分钟在SS-34或8,000rpm×20分钟内的颗粒细胞(约8,000xg时为10-20分钟) em>)。
  2. 浮游生物
    1. 将缓冲液A中的10ml(2×50ml培养物)或100ml(3×700ml培养物)的RT悬浮细胞沉淀(参见方案1)。
    2. 加入100μg/ml溶菌酶(10mg/ml储备溶液),并在室温下孵育,直到细胞变得渗透(不要长于需要处理细胞) 5-10分钟为此,进行渗透敏感性试验。将50μl细胞置于玻璃管中的1 ml 10 mM EDTA,pH 7.0中。观察到的裂解(悬浮液变得透明)应该在1-2分钟内发生。在这些条件下,没有溶菌酶的细胞不会被破坏 注意:记住在溶菌酶添加之前要控制!没有溶菌酶的细胞保持完整,应与溶菌酶处理的细胞进行比较,通过匹配细胞悬浮液的浊度来进行比较。
    3. 加入4mM MgSO 4(来自1M储备溶液)和痕量DNA酶。悬浮液粘度几乎会立即下降。
    4. 在SS-34中以7,500×g(8,000rpm)×10分钟的颗粒状原生质球。
    5. 将冰原上的原生质球悬浮于20毫升或40毫升冷缓冲液B中(见方案2)。
    6. 加入2mM二硫苏糖醇(DTT)(来自100mM储备溶液),0.5mM PMSF(来自100mM新鲜储备液)。
      注意:
      1. PMSF在水溶液中使用寿命短。只有在无水乙醇保持厌氧的情况下才是稳定的,所以最好的方法是在95%的乙醇中制备新鲜的储液。
      2. 该培养基是任选的,可以依赖于进一步的实验而改变,然而,高的[Mg 2+],二硫苏糖醇(DTT)和蛋白酶抑制剂PMSF总是显着改善膜质量。缓冲液B被开发用于通过质子泵测量囊内酸化。为了监测低盐浓度的电位产生缓冲液C(见下文)。
  3. 膜(冰/水浴)
    1. 超声处理的原生质球在约75 W(瓦特)。为了有效的超声处理,超声波发生器尖端和具有原生质球悬液的管壁之间的距离不应大于6-8mm。为了防止过热,使用50%超声波脉冲。大约超声处理30秒,冷却1分钟。对于渗透敏感细胞,应重复4-6次。细胞破坏可以被视觉控制。
    2. 在SS-34中以7,200×g×10分钟的颗粒未破裂的细胞和细胞碎片,并将其丢弃。
    3. 30,000 x g(40,000 rpm)x 40 min的Ti70中的颗粒膜或80,000-150,000 x g的其他合适的转子30-40分钟。如果需要较大内部体积的膜囊泡,则沉降应进行更短或较低的RCF(相对离心力)。
    4. 如果需要,冲洗沉淀物,并悬浮所需缓冲液B的膜(最小体积)(参见方法2)。
    5. 如果不使用膜,立即将其以等分试样冷冻在液体N 2中,并储存在-80℃。
    6. 通过使用ψ敏感探针Oxonol VI监测由质子泵产生的电位(ψΔψ),可以简单地测试获得的膜囊泡的质量,如图1。可以通过用施加的扩散电位进行吸收变化的校准来估计Δψ的值,例如,Gulli等人,1997; Pouliquin 等人,1999)。可以在质子解偶联剂如羰基氰化物间氯苯基肼(CCCP)或钾转运蛋白缬氨霉素存在下建立扩散电位。可以通过能斯特方程计算扩散电位的值。该程序的详细描述可以在Gulli等人,1997年的论文中找到。
    7. 膜结合复合物I的NADH:泛醌还原酶活性可以在缓冲液C中在30℃下通过在340nm处进行NADH氧化来测量(ε= 6.2mM /SUP>)。由E,E制备的膜泡囊中的复合物I的NADH:泛醌氧化还原酶活性。大肠杆菌GR70N为0.92±0.13(n = 10)(Belevich等人,2011)。


      图1.在E中的三个质子泵产生电势之后。使用Δψ敏感性探针Oxonol VI进行大肠杆菌膜。将膜泡囊装载缓冲液D.该培养基由用5mM(NH 3) (将ΔpH转换为Δψ)和Oxonol VI2.5μM(存储在2-5中) mM乙醇或甲醇储备溶液,-20℃,稳定至少一半)。以80μg/ml的浓度加入膜。反应之后是如所示的吸收变化的测量。通过NADH:泛素氧化还原酶I型(复合体I)监测Δψ
      产物,用100μM十二氟酮和1mM KCN完成培养基以阻断末端氧化酶。加入50μMNADH以开始反应,由浓度1μM的复合物I抑制剂,罗替尼汀(Rollini)消散Δψ。通过喹诺酮氧化酶 3 监测Δψ,使用50μM泛醌1完成培养基。加入反应物2mM二硫苏糖醇,通过1mM KCN加入消散Δψ。为了通过ATPase监测产生Δψ,用1mM MgSO 4完成培养基。通过加入1mM ATP开始反应,通过加入浓度为10μM的ATP酶抑制剂,羟化蛋白B(Auro B)消耗Δψ。

笔记

  1. 该协议提供了高度可重复的结果,但是所有程序应尽可能快地进行。断裂(特别是在细胞破碎步骤之后)可能导致通过膜的质子泄漏增加,膜酶活性降低。
  2. 该协议特别为E开发。大肠杆菌细胞,不应该用于其他细菌物种而不进行修改。

食谱

  1. 缓冲区A
    200mM Tris-HCl,pH8.0
    2 mM EDTA
    30%蔗糖
  2. 缓冲区B
    100mM HEPES-KOH,pH7.5
    100 mM KCl
    10mM MgCl 2
  3. 缓冲区C
    25mM HEPES-BTP,pH7.5
    3 mM KCl
  4. 缓冲区D
    100mM MOPS-BTP,pH 7.0
    1mM MgSO 4

致谢

这项工作得到了Biocentrum赫尔辛基,Sigrid Juselius基金会和芬兰科学院的资助。

参考

  1. Euro,L.,Belevich,G.,Verkhovsky,M.I.,Wikström,M.和Verkhovskaya。 M.(2008)。保留的膜的赖氨酸残基亚基NuoM通过质子泵浦的NADH:泛醌氧化还原酶(复合物I)参与能量转换。 Biochim Biophys Acta 1777(9):1166-1172。
  2. Belevich,G.,Knuuti,J.,Verkhovsky,MI,Wikström,M,Verkhovskaya,M。(2011)。  通过定点突变检测大肠杆菌呼吸复合物I的亚单位L中的长α螺旋的机械作用。 Mol Microbiol 82(5):1086-1095。
  3. Ghelli A.,Benelli,B.和Esposti,MD(1997)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/?term=+的+膜+电位的测量+++由复杂+ I+中+ submitochondrial颗粒+生成+"。 target ="_ blank">测定由复合物I在遗传性粒子中产生的膜电位。生物化学121(4):746-755。
  4. Pouliquin,P.,Grouzis,J.和Gibrat,R。(1999)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/?term通过与氧杂菁++ VI +++被动NO3-+传输+的电=+研究+++分离植物根+++血浆膜"。 目标="_ blank">通过被分离的植物根膜进行被动NO 3转运的oxonol VI的电生理研究。 Biophys J > 76:360-373。
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引用:Verkhovskaya, M. (2017). Preparation of Everted Membrane Vesicles from Escherichia coli Cells. Bio-protocol 7(9): e2254. DOI: 10.21769/BioProtoc.2254.
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