搜索

Measurement of Proton-driven Antiport in Escherichia coli
大肠杆菌质子逆向运输蛋白活性的测定   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Secondary active transport of substrates across the inner membrane is vital to the bacterial cell. Of the secondary active transporter families, the ubiquitous major facilitator superfamily (MFS) is the largest and most functionally diverse (Reddy et al., 2012). Recently, it was reported that the MFS multidrug efflux protein MdtM from Escherichia coli (E. coli) functions physiologically in protection of bacterial cells against bile salts (Paul et al., 2014). The MdtM transporter imparts bile salt resistance to the bacterial cell by coupling the exchange of external protons (H+) to the efflux of bile salts from the cell interior via an antiport reaction. This protocol describes, using fluorometry, how to detect the bile salt/H+ antiport activity of MdtM in inverted membrane vesicles of an antiporter-deficient strain of E. coli TO114 cells by measuring transmembrane ∆pH. This method exploits the changes that occur in the intensity of the fluorescence signal (quenching and dequenching) of the pH-sensitive dye acridine orange in response to changes in [H+] in the vesicular lumen. Due to low levels of endogenous transporter expression that would normally make the contribution of individual transporters such as MdtM to proton-driven antiport difficult to detect, the method typically necessitates that the transporter of interest be overexpressed from a multicopy plasmid. Although the first section of the protocol described here is very specific to the overexpression of MdtM from the pBAD/Myc-His A expression vector, the protocol describing the subsequent measurement of bile salt efflux by MdtM can be readily adapted for measurement of antiport of other substrates by any other antiporter that exchanges protons for countersubstrate.

Materials and Reagents

  1. pBAD/Myc-His A expression vector (Life Technologies, catalog number: V440-01 )
  2. L-(+)-arabinose (Sigma-Aldrich, catalog number: A3256 )
  3. Carbenicillin (Carbenicillin Direct)
  4. Agar (Sigma-Aldrich, catalog number: A1296 )
  5. Tryptone (Fluka, catalog number: T7293 )
  6. Yeast extract (Fluka, catalog number: 92114 )
  7. Potassium chloride (Thermo Fisher Scientific, catalog number: BP366 )
  8. Escherichia coli (E. coli) TO114 (gift of Prof. Hiroshi Kobayashi, Chiba University, Japan)
  9. Acridine orange hemi (zinc chloride) salt (Sigma-Aldrich, catalog number: A6014 )
  10. BisTris propane (BTP) (Sigma-Aldrich, catalog number: B6755 )
  11. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  12. Deoxyribonuclease I (DNase) from bovine pancreas (Sigma-Aldrich, catalog number: DN25 )
  13. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Sigma-Aldrich, catalog number: C2759 )
  14. Sodium DL-lactate solution 50% aqueous (VWR International, catalog number: 27927.298 )
  15. Magnesium sulphate heptahydrate (MgSO4.7H2O) (Thermo Fisher Scientific, catalog number: M/1000/60 )
  16. Sodium cholate hydrate (Sigma-Aldrich, catalog number: C1254 )
  17. Absolute ethanol (Thermo Fisher Scientific, catalog number: E/0650DF/17 )
  18. High purity (18 MΩ) Millipore or AnalR water
  19. Choline chloride (Sigma-Aldrich, catalog number: C1879 )
  20. Sucrose (Sigma-Aldrich, catalog number: 84097 )
  21. DL-dithiothreitol (Sigma-Aldrich, catalog number: 43815 )
  22. TRIZMA base (Sigma-Aldrich, catalog number: T1503 )
  23. 32% hydrochloric acid (VWR International, catalog number: 20254.321 )
  24. LBK agar (see Recipes)
  25. LBK liquid medium (see Recipes)
  26. Tris/choline/dithiothreitol/sucrose (TCDS) buffer (see Recipes)
  27. Transport assay buffer (see Recipes)
  28. 200 mM sodium DL-lactate solution (see Recipes)

Equipment

  1. Temperature-controlled shaking incubator for bacterial growth
  2. Petri dishes for bacterial colony growth
  3. 100 ml conical flasks
  4. 250 ml conical flasks (x2)
  5. 5,000 ml conical flasks (x2)
  6. Large ice bucket
  7. Refrigerated, large capacity centrifuge and rotor for harvesting bacterial cells
  8. 1,000 ml or 500 ml centrifuge pots and lids
  9. Benchtop vortexer
  10. 100 ml beaker and stir bar to fit
  11. Magnetic stirrer
  12. 25 ml disposable plastic pipettes
  13. Selection of single channel pipettes (1,000 µl, 100 µl, 20 µl, 10 µl)
  14. Pipette tips for above
  15. Refrigerated centrifuge and rotor capable of spinning ~50 ml tubes at 18k x g
  16. Refrigerated ultracentrifuge, rotor and polycarbonate ultracentrifuge tubes capable of handling ~30 - 50 ml volumes
  17. French Press (Thermo Fisher Scientific, catalogue number: FA-078 )
  18. Standard pressure cell (40 kpsi; 35 ml capacity) (Thermo Fisher Scientific, catalog number: FA-031 )
  19. 50 ml syringes (for filtering solutions)
  20. 0.22 µm sterile filters to fit 50 ml syringe
  21. 1 L and 500 ml Duran bottles with lids
  22. 1.5 ml Eppendorf tubes
  23. Medical wipes (Kimwipes®)
  24. Parafilm
  25. UV/vis spectrophotometer and 10 mm pathlength quartz cuvette
  26. 10 x 4 mm, 1,400 µl volume quartz cuvette for fluorescence spectroscopy (Hellma, catalog number: 104F-QG )
  27. Small magnetic stir bar to fit inside quartz cuvette
  28. Fluorometer e.g. Fluoromax-4 (Horiba) capable of performing time-based acquisition measurements and fitted with a temperature controlled cuvette holder and stirrer
  29. Source of compressed air (either aerosol can or fixed supply) for drying cuvette

Procedure

  1. Growth and harvesting of bacterial cultures and preparation of inverted membrane vesicles
    1. Perform a fresh transformation with the pBAD/Myc-His A plasmid DNA encoding wild type or mutant transporter (or your vector of choice encoding your transporter of interest) into chemically competent E. coli TO114 cells. Plate the cells onto Luria Bertani Potassium (LBK) agar containing 100 μg/ml carbenicillin for selection. LBK agar must be used instead of regular Luria Bertani (LB) agar because the latter contains NaCl and the TO114 strain is sensitive to this salt due to deletion of the chromosomally encoded Na+/H+ antiporter NhaA. Incubate the plate overnight (~15 to ~18 h) at 37 °C.
    2. Pick a few single colonies from the agar plate and use to inoculate 2 x 250 ml conical flasks, each containing 100 ml of LBK liquid medium supplemented with 100 μg/ml carbenicillin. Grow the cultures overnight (~15 h) at 30 °C with 250 rpm shaking in a temperature-controlled shaking incubator. Measure the optical density at 600 nm (OD600) of the culture using a spectrophotometer; it should be between 3.0-3.3. If the OD600 is less than 3.0, then incubate for a further 0.5-1.0 h and re-measure. If the OD600 is greater than 3.3, the cultures have overgrown and fresh cultures will need to be prepared.
    3. Inoculate 2 x 5 L conical flasks, each containing 1,000 ml of LBK liquid medium supplemented with 100 μg/ml carbenicillin, with 15 ml of overnight culture. Incubate at 32 °C with 270 rpm shaking for ~2.5 h in a temperature-controlled shaking incubator. The OD600 should be ~0.6. Drop the temperature to 25 °C and grow with 270 rpm shaking until the OD600 is 1.0. This usually takes between 0.5 to 1.0 h.
    4. Induce overexpression of MdtM by addition of 0.1% w/v L-(+)-arabinose [5 ml of 20% w/v L-(+)-arabinose] to each flask. After addition of arabinose, grow the cells for a further 1.5 h at 25 °C with 270 rpm shaking prior to harvesting.
    5. Transfer the E. coli TO114 cells that contain overexpressed transporter into 500 ml or 1,000 ml capacity centrifuge pots that have been pre-chilled on ice for at least 15 min. Pre-cool the centrifuge to 4 °C and harvest cells by centrifugation at 5,000 x g for 20 min.
    6. Decant the supernatant and wash the pelleted cells by resuspending in chilled (4 °C) TCDS buffer. Use 30 ml of TCDS buffer for each one litre of cell culture that was pelleted. Maintain the cells on ice during this procedure. Harvest the washed cells by centrifugation as described in step A1 (above) and repeat the washing procedure. Resuspend the resultant cell pellet in 30 ml of chilled TCDS buffer containing 2 mM PMSF (which should be made up as a 100 mM stock in ethanol and stored at -20 °C; ensure the solution is thawed thoroughly and vortexed vigorously before use) and 5 µM DNase and maintain the mixture on ice.
      Notes:
      1. Typically, a 1 L culture of bacterial cells will provide sufficient material for these experiments.
      2.  Cells should be resuspended either by gentle vortexing using a benchtop vortexer or by gentle aspiration using a 25 ml sterile plastic pipette.
    7. Decant the resuspended cells into a 100 ml beaker containing a stir bar, place on magnetic stirrer and stir in a cold room at 4 °C for 20 min. Alternatively, place the beaker on ice in an ice bucket, place on the magnetic stirrer and stir for 20 min.
    8. The method for production of inverted vesicles relies on a combination of the fluid shear forces and decompression created as the cell mixture passes through the needle valve of a French pressure cell. Generate inverted membrane vesicles by a single passage of the resuspended cell mixture through a French pressure cell at a minimum of 4,000 psi. If the pressure is too low, inverted vesicles will not be formed. The pressure cell should be chilled on ice for ~30 min prior to use. The resulting inverted vesicle mixture should be collected in a 100 ml conical flask kept on ice.
    9. Decant the mixture into a pre-chilled ~50 ml centrifuge tube and remove any unbroken cells and cell debris by centrifugation at 18,000 x g for 10 min at 4 °C. Carefully decant the supernatant (do not disturb the pellet that contains unbroken cells and cell debris) containing the cell membrane vesicles into a pre-chilled 30-50 ml volume polycarbonate ultracentrifuge tube on ice.
    10.  Harvest the inverted vesicles by ultracentrifugation at 100,000 x g for 1 h at 4 °C. Carefully decant the supernatant and retain the pellet. Place the ultracentrifuge tube containing the pelleted vesicles on ice.
    11. Resuspend the inverted vesicle pellet in 1 ml of ice-cold TCDS buffer by gentle aspiration using a 1,000 µl pipette. Transfer the resuspended vesicles to a pre-chilled 1.5 ml Eppendorf tube on ice for use in the transport assay. In our experience, vesicles stored on ice are stable for several hours.
    12. Quantify the total membrane protein content of the inverted vesicles by UV absorbance spectroscopy at 280 nm. Blank the spectrophotometer using a 10 mm pathlength quartz cuvette containing 1,000 µl of TCDS buffer. The buffer should be at room temperature to prevent frosting of the cuvette faces. Clean the faces of the cuvette using a fresh paper wipe prior to measurement. Once the spectrophotometer is blanked, remove 5 µl of buffer from the cuvette using a 10 µl pipette and replace with 5 µl of vesicles. Cover the opening of the cuvette with a square of Parafilm and invert the cuvette a few times to ensure a homogeneous distribution of vesicles. Record the absorbance of the vesicle mixture at 280 nm and calculate the total membrane protein concentration assuming that an A280 of 1.0 is equivalent to a protein concentration of 1.0 mg/ml.
      Notes:
      1. Remember to multiply the 280 nm absorbance value you obtain by a factor of 200 to calculate the concentration of the undiluted vesicle mixture in mg/ml.
      2. At this stage of the preparation, the resuspended vesicles can be transferred to tubes in aliquots of 25-100 µl, snap-frozen in liquid nitrogen and stored at -80 °C for subsequent use. Vesicles frozen in this way will retain their integrity for several months. However, if frozen vesicle stocks are used for the subsequent transport measurements, the vesicles must be thawed very slowly on ice prior to use to prevent their fracture.

  2. Fluorometric antiport assays
    1. In this section we describe the set-up parameters for a Fluoromax-4 fluorometer. These parameters, however, can form the basis for the set-up of other fluorometers. Once the instrument is switched on and the software booted up, set the temperature of the cuvette holder to 25 °C. Open the instrument software and select for time-based data acquisition with excitation and emission wavelengths of 492 nm and 525 nm, respectively. Set the excitation and emission slit widths to 1.5 nm and 2.5 nm, respectively.
    2. Add an aliquot of inverted vesicles (which should be maintained on ice in TCDS buffer) to room temperature transport assay buffer containing the acridine orange probe in a 10 mm x 4 mm quartz cuvette to a final concentration of 0.5 mg/ml membrane protein in a total volume of 1,500 µl. The longest pathlength of the cuvette should face the excitation light source. Place a small magnetic flea into the cuvette and stir the contents gently. Allow the vesicles and assay buffer to equilibrate for ~200 sec.
    3. Start recording the fluorescence emission. After approximately 50 sec, add 15 µl of 200 mM stock sodium DL-lactate solution to the cuvette contents to give a final sodium DL-lactate concentration of ~2.0 mM. Addition of lactate energises the vesicles and generates a respiration dependent ΔpH (acid inside) across the inverted vesicle membrane as H+ is pumped into the vesicle interior. This causes a dequench of the acridine orange fluorescence signal (see Figure 1in Representative data).
    4. Following the establishment of a ΔpH, monitor the acridine orange fluorescence dequench for a further ~200 sec until it stabilises. Initiate MdtM-mediated, proton-driven antiport by adding substrate (in this case the bile salt sodium cholate) to the inverted vesicle mixture. We added 12.5 µl of 250 mM stock solution made up in high purity water to give a final concentration of sodium cholate in the cuvette of ~2.0 mM. For other substrates we suggest testing a range varying from 1 mM to 100 mM to establish the concentration that gives the best dequench signal. Upon addition of substrate, there should be an immediate dequench (rise) of the acridine orange fluorescence emission signal (see Figure 1a) due to dissipation of the established ΔpH as a result of MdtM-mediated sodium cholate/H+ antiport activity and concomitant alkalinisation of the vesicle lumen.
    5. Record the fluorescence dequench signal for 50 sec to allow the antiport reaction to achieve a steady state (as observed by a plateauing of the fluorescence signal). Addition of the protonophore CCCP to a final concentration of ~100 µM (1.6 µl of a 100 mM stock made up in ethanol) in the assay mixture should be performed to abolish ΔpH-driven, MdtM-mediated antiport activity. Addition of CCCP will cause a further dequench of the fluorescence signal as the ΔpH is dissipated (see Figure 1). Record the fluorescence signal for a further ~50 sec then terminate the acquisition and save the electronic data.
    6. Decant the cuvette contents into a suitable waste container and wash the cuvette thoroughly with ethanol then high-purity water. Dry the cuvette carefully with compressed air.

Representative data


Figure 1. Representative measurements of the fluorescence quench/dequench of acridine orange upon addition of bile salts to inverted vesicles of E. coli TO114 cells that overproduced recombinant (a) wild type MdtM or, as a control, (b) the dysfunctional MdtM D22A mutant. Respiration-dependent generation of ΔpH (acid inside) was established by addition of sodium DL-lactate as indicated. Sodium cholate was added to vesicles as indicated to initiate the transport reaction and CCCP was used to dissipate ΔpH. The fluorescence dequench observed in the control experiment (panel b) upon addition of sodium cholate is due to antiport activity of chromosomally encoded MdtM. Fluorescence intensity is measured in counts per second (cps). Note that the fluorescence intensity you measure may differ depending upon how your instrument is set up. The traces above are representative of experiments performed in triplicate on at least two separate preparations of inverted vesicles.

Notes

  1. To ensure reproducibility, the assays should be performed in triplicate on at least two separate preparations of inverted vesicles.
  2. If the fluorescence signal does not quench, or enhances, the vesicles have either not maintained integrity or are not inverted and their preparation needs to be repeated.
  3. As with all assays that rely on detection of fluorescence, robust controls must be in place to ensure that any detected transport activity can be attributed unambiguously to the protein of interest. In our experiments, we used inverted vesicles that overexpressed MdtM D22A, a dysfunctional point mutant of MdtM, as a negative control (see Figure 1b).
  4. If the method is to be used for detection of metal ion/H+ antiport activity, the use of inverted vesicles generated from the antiporter-deficient TO114 strain of E. coli is important because at least four other transporters (NhaA, NhaB, ChaA and MdfA) present in the bacterium catalyse a monovalent metal cation/H+ exchange.
  5. Finally, if this protocol is used for comparison of antiport activities of wild type and mutant transporters, the amount of target protein present in the inverted vesicle membranes must be quantified (usually by immunodetection methods) to ensure that any measured differences in H+ uptake are due solely to differences in the activity of the transporters and not to differences in expression levels.

Recipes

  1. LBK agar (100 ml)
    1.0 g tryptone
    0.5 g yeast extract
    0.745 g KCl
    1.5 g agar
    Make up to 100 ml with high purity water then autoclave
    Add 100 μg/ml carbenicillin for selection when the solution is still liquid and warm to the touch
    Add to Petri dishes under sterile conditions
  2. LBK liquid medium (1 L)
    10 g tryptone
    5 g yeast extract
    7.45 g KCl
    Make up to 1,000 ml with high purity water then autoclave
  3. Tris/choline/dithiothreitol/sucrose (TCDS) buffer (1 L)
    Consisting of 10 mM Tris-HCl (pH 7.5), 140 mM choline chloride, 0.5 mM dithiothreitol and 250 mM sucrose
    1 M Tris-HCl (pH 7.5) 10 ml
    2 M choline chloride 70 ml
    1 M sucrose 250 ml
    Make up to 999.5 ml with high purity water then check pH and adjust if necessary with HCl
    Autoclave or sterile filter and store at 4 °C
    Add 0.5 ml dithiothreitol from frozen 1 M stocks immediately before use
  4. Transport assay buffer (100 ml)
    Consisting of 10 mM BisTris propane (pH 7.2), 5 mM MgSO4, 1 µM acridine orange
    100 mM BisTris propane (pH 7.2) 10 ml
    2 M MgSO4 0.25 ml
    10 mM acridine orange 10 µl
    Make up to 100 ml with high purity water then sterile filter
    Acridine orange is light sensitive so both the transport assay buffer and acridine orange stocks should be stored either in amber bottles or in a container protected from light at 4 °C in the dark
  5. 200 mM sodium DL-lactate solution (10 ml)
    0.45 ml 50% sodium DL-lactate aqueous solution
    Make up to 10 ml with high-purity water
    Sterile filter and stored at 4 °C

Acknowledgments

    This work was supported by BBSRC grant BB/K014226/1 (to CJL). The protocol described above is adapted from one reported previously (Resch et al., 2010).

References

  1. Paul, S., Alegre, K. O., Holdsworth, S. R., Rice, M., Brown, J. A., McVeigh, P., Kelly, S. M. and Law, C. J. (2014). A single-component multidrug transporter of the major facilitator superfamily is part of a network that protects Escherichia coli from bile salt stress. Mol Microbiol 92(4): 872-884.
  2. Reddy, V. S., Shlykov, M. A., Castillo, R., Sun, E. I. and Saier, M. H., Jr. (2012). The major facilitator superfamily (MFS) revisited. FEBS J 279(11): 2022-2035.
  3. Resch, C. T., Winogrodzki, J. L., Patterson, C. T., Lind, E. J., Quinn, M. J., Dibrov, P. and Hase, C. C. (2010). The putative Na+/H+ antiporter of Vibrio cholerae, Vc-NhaP2, mediates the specific K+/H+ exchange in vivo. Biochemistry 49(11): 2520-2528.

简介

底物穿过内膜的二次主动转运对细菌细胞是至关重要的。在次要活性转运蛋白家族中,遍在的主要促进子超家族(MFS)是最大和最功能多样的(Reddy等人,2012)。最近,据报道,来自大肠杆菌(大肠杆菌)的MFS多药物外排蛋白MdtM在保护细菌细胞对抗胆汁盐中具有生理功能(Paul等人al。,2014)。 MdtM转运蛋白通过将外部质子(H + +)的交换耦合到通过反向端反应从细胞内部排出的胆汁盐,赋予细菌细胞以抗胆汁盐性。该方案使用荧光测定法描述了如何在E的逆向转运体缺陷菌株的倒置膜囊泡中检测MdtM的胆汁盐/H sup/+抗末端活性。通过测量跨膜ΔpH测定大肠杆菌 TO114细胞。该方法利用pH敏感染料吖啶橙的荧光信号(淬灭和去淬灭)的强度响应于囊泡腔中的[H sup +]的变化而发生的变化。由于内源性转运蛋白表达的低水平,其通常使得单个转运蛋白例如MdtM对质子驱动的反运输蛋白的贡献难以检测,所述方法通常需要从多拷贝质粒过表达所关注的转运蛋白。尽管本文所述的方案的第一部分对于来自pBAD/emyc-His-A表达载体的MdtM的过表达非常特异,但描述随后通过MdtM测量胆汁盐流出的方案可以容易地适用于通过任何其他反交换器测量其它底物的反向运输,其交换质子用于对衬底。

材料和试剂

  1. pBAD/myc -His表达载体(Life Technologies,目录号:V440-01)
  2. L - (+) - 阿拉伯糖(Sigma-Aldrich,目录号:A3256)
  3. 羧苄青霉素(Carbenicillin Direct)
  4. 琼脂(Sigma-Aldrich,目录号:A1296)
  5. 胰蛋白胨(Fluka,目录号:T7293)
  6. 酵母提取物(Fluka,目录号:92114)
  7. 氯化钾(Thermo Fisher Scientific,目录号:BP366)
  8. 大肠杆菌(大肠杆菌)TO114(日本千叶大学Kobayashi教授的礼物)
  9. 吖啶橙半(氯化锌)盐(Sigma-Aldrich,目录号:A6014)
  10. BisTris丙烷(BTP)(Sigma-Aldrich,目录号:B6755)
  11. 苯基甲磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)
  12. 来自牛胰腺的脱氧核糖核酸酶I(DNase)(Sigma-Aldrich,目录号:DN25)
  13. 羰基氰化物3-氯苯基腙(CCCP)(Sigma-Aldrich,目录号:C2759)
  14. DL-乳酸钠溶液50%水溶液(VWR International,目录号:27927.298)
  15. 硫酸镁七水合物(MgSO 4·7H 2 O 7H 2 O)(Thermo Fisher Scientific,目录号:M/1000/60)
  16. 胆酸钠水合物(Sigma-Aldrich,目录号:C1254)
  17. 无水乙醇(Thermo Fisher Scientific,目录号:E/0650DF/17)
  18. 高纯度(18MΩ)Millipore或AnalR水
  19. 氯化胆碱(Sigma-Aldrich,目录号:C1879)
  20. 蔗糖(Sigma-Aldrich,目录号:84097)
  21. DL二硫苏糖醇(Sigma-Aldrich,目录号:43815)
  22. TRIZMA碱(Sigma-Aldrich,目录号:T1503)
  23. 32%盐酸(VWR International,目录号:20254.321)
  24. LBK琼脂(见配方)
  25. LBK液体介质(见配方)
  26. Tris /胆碱/二硫苏糖醇/蔗糖(TCDS)缓冲液(参见Recipes)
  27. 运输测定缓冲液(参见配方)
  28. 200 mM DL-乳酸钠溶液(见配方)

设备

  1. 温控的细菌生长摇动培养箱
  2. 用于细菌菌落生长的培养皿
  3. 100ml锥形瓶
  4. 250ml锥形瓶(×2)
  5. 5,000ml锥形瓶(×2)
  6. 大冰桶
  7. 冷冻,大容量离心机和转子收获细菌细胞
  8. 1,000 ml或500 ml离心机罐和盖
  9. 台式涡流器
  10. 100毫升烧杯和搅拌棒以适应
  11. 磁力搅拌器
  12. 25 ml一次性塑料移液器
  13. 选择单通道移液器(1,000μl,100μl,20μl,10μl)
  14. 以上的移液器提示
  15. 冷冻离心机和转子,能够以18k/g 旋转约50ml管子
  16. 冷冻超速离心机,转子和聚碳酸酯超速离心管,能够处理〜30 - 50 ml的体积
  17. French Press(Thermo Fisher Scientific,目录号:FA-078)
  18. 标准压力室(40kpsi; 35ml容量)(Thermo Fisher Scientific,目录号:FA-031)
  19. 50 ml注射器(用于过滤溶液)
  20. 0.22μm无菌过滤器,适合50 ml注射器
  21. 1升和500毫升杜兰瓶盖子
  22. 1.5 ml Eppendorf管
  23. 医用湿巾(Kimwipes ®
  24. parafilm
  25. UV/vis分光光度计和10mm长度石英比色皿
  26. 10×4mm,用于荧光光谱的1400μl体积石英比色杯(Hellma,目录号:104F-QG)
  27. 小型磁力搅拌棒,以适应石英比色杯
  28. 荧光计。 Fluoromax-4(Horiba),可进行基于时间的采集测量,并配有温度受控的试管架和搅拌器
  29. 用于干燥比色皿的压缩空气源(气溶胶罐或固定供应源)

程序

  1. 细菌培养物的生长和收获和倒置膜囊泡的制备
    1. 用pBAD/emc -His A质粒DNA进行新的转化 编码野生型或突变型转运蛋白(或您的选择的载体 编码您感兴趣的转运蛋白)转化为化学感受态。大肠杆菌 TO114细胞。将细胞铺在Luria Bertani钾(LBK)琼脂上 含有100μg/ml羧苄青霉素用于选择。必须使用LBK琼脂 而不是常规的Luria Bertani(LB)琼脂,因为后者包含 NaCl和TO114菌株由于缺失对该盐敏感 染色体编码的Na +/+/H + +反向转运体NhaA。孵育平板 在37℃过夜(〜15至约18小时)
    2. 从中选择几个单一的殖民地  琼脂板上并用于接种2×250ml锥形瓶 含有100ml补充有100μg/ml的LBK液体培养基 羧苄青霉素。在30℃下以250rpm生长培养物过夜(〜15h)  在温度控制的摇动培养箱中振荡。测量 使用a。在600nm处的光密度(OD <600) 分光光度计;它应该在3.0-3.3之间。如果OD <600> 较小 比3.0,然后再孵育0.5-1.0小时并重新测量。如果 OD 600大于3.3,培养物长满和新鲜 文化将需要准备
    3. 接种2 x 5 L锥形 烧瓶中,每个含有1,000ml补充有LBK液体培养基 100μg/ml羧苄青霉素,15ml过夜培养物。 在32℃孵育   ℃,在温度控制的摇动中以270rpm摇动约2.5小时 孵化器。 OD <600> 应为〜0.6。 将温度降至25°C和 以270rpm摇动生长直至OD 600为1.0。 这通常需要 在0.5至1.0小时之间
    4. 通过加入诱导MdtM的过表达   0.1%w/v L - (+) - 阿拉伯糖[5ml 20%w/v L - (+) - 阿拉伯糖] 烧瓶。 加入阿拉伯糖后,将细胞再培养1.5小时 在25℃,270rpm摇动,然后收获
    5. 传输 E。   大肠杆菌 TO114细胞,其含有过表达的转运蛋白到500ml或 1,000毫升容量离心机罐已经预先在冰上冷冻 至少15分钟。预冷离心机至4°C并收获细胞 在5,000xg离心20分钟。
    6. 滗析上清液 并通过在冷冻(4℃)TCDS中重悬来洗涤沉淀的细胞 缓冲。使用30毫升TCDS缓冲液每升一升的细胞培养物  沉淀。在此过程中保持细胞在冰上。收成 通过如步骤A1(上述)中所述的离心来洗涤细胞 重复洗涤程序。在30中重悬所得细胞沉淀 ml含有2mM PMSF的冷冻TCDS缓冲液(其应该补充 作为100mM储备在乙醇中并储存在-20℃;确保解决方案 在使用前彻底解冻并剧烈涡旋)和5μMDNA酶 并将混合物保持在冰上
      1. 通常,1L细菌细胞培养物将为这些实验提供足够的材料。
      2.  细胞应通过使用台式轻轻涡旋重悬   涡旋器或通过使用25ml无菌塑料移液管温和抽吸。
    7. 将重悬的细胞倒入含有搅拌器的100ml烧杯中 棒,置于磁力搅拌器上并在4℃的冷室中搅拌20分钟 min。 或者,将烧杯置于冰上的冰桶中,放置 磁搅拌器并搅拌20分钟
    8. 方法为 反向囊泡的产生依赖于流体的组合 剪切力和当细胞混合物通过时产生的减压 通过法国压力单元的针阀。 生成反转 通过重悬的细胞混合物的单次通过来分离膜囊泡 通过法式压力室至少4,000psi。 如果 压力太低,不会形成反相囊泡。 压力 细胞应在冰上冷冻〜30分钟后使用。 所结果的 倒置的囊泡混合物应收集在100ml锥形瓶中 保持在冰上
    9. 将混合物倒入预冷的〜50ml 离心管并除去任何未破损的细胞和细胞碎片 在4℃下以18,000×g离心10分钟。 小心倾倒 上清液(不要打扰含有未破坏细胞的沉淀物 细胞碎片)包含在预冷冻的细胞膜囊泡中 在冰上用30-50ml体积的聚碳酸酯超速离心管
    10.  通过超速离心以100,000 x g 收获倒置的囊泡1分钟   h。 小心倾析上清液并保留沉淀。 将含有沉淀囊泡的超离心管置于冰上
    11. 重悬在1ml冰冷的TCDS缓冲液中倒置的囊泡沉淀 通过使用1000μl移液管温和吸出。转移重悬 小泡置于预冷的1.5ml Eppendorf管中,用于冰上 运输测定。根据我们的经验,存储在冰上的泡囊是稳定的 几个小时。
    12. 量化总膜蛋白含量  通过在280nm处的UV吸收光谱测定倒置的囊泡。空白 分光光度计使用10 mm光程石英比色杯  1,000μlTCDS缓冲液。缓冲液应在室温至室温 防止反应杯面的结霜。清洁试管的表面 在测量前使用新鲜的纸巾。一旦 分光光度计为空白,从比色杯中取出5μl缓冲液 使用10μl移液管并用5μl的囊泡替换。封面 打开具有Parafilm正方形的比色皿并倒置比色皿a  几次以确保囊泡的均匀分布。记录 在280nm处的囊泡混合物的吸光度并计算总量 膜蛋白浓度,假设A 280为1.0 相当于蛋白质浓度为1.0mg/ml 注意:
      1. 记住乘以一个因子获得的280 nm吸光度值 为200,计算未稀释的囊泡混合物的浓度 以mg/ml为单位。
      2. 在这个准备阶段,重新悬浮 囊泡可以以25-100μl的等分试样转移到管中, 在液氮中快速冷冻并储存在-80℃下用于随后的使用。 以这种方式冻结的囊泡将保持其完整性几个 个月。 然而,如果冷冻的囊泡原液用于随后的 运输测量,囊泡必须在冰上非常缓慢地解冻 在使用前防止其骨折。

  2. 荧光法测定
    1. 在本节中,我们描述了Fluoromax-4的设置参数 荧光计。 然而,这些参数可以形成基础 设置其他荧光计。 一旦仪器开启,   软件启动,将试管架的温度设置为25°C。   打开仪器软件并选择基于时间的数据采集   激发和发射波长为492nm和525nm, 分别。 设置激发和发射缝隙宽度为1.5 nm和 2.5nm。
    2. 添加一等分的倒囊泡( 应在TCDS缓冲液中的冰上保持)至室温 运输测定缓冲液中含有吖啶橙探针在10mm x  4mm石英比色皿中至终浓度为0.5mg/ml膜 蛋白质总体积为1500μl。最长路径长度 比色杯应面向激发光源。放一个小磁 跳蚤进入比色杯,轻轻搅拌内容物。允许囊泡 和测定缓冲液平衡〜200秒
    3. 开始录制 荧光发射。大约50秒后,加入15μl的200 mM 储备钠DL-乳酸盐溶液到比色皿内容物以给出最终  DL-乳酸钠浓度为〜2.0mM。加入乳酸盐 激发囊泡并产生呼吸依赖性ΔpH(酸 内)穿过倒置的囊泡膜,当H sup + i被泵入 囊泡内部。这导致吖啶橙的脱猝灭 荧光信号(见代表数据中的图1)
    4. 在建立ΔpH后,监测吖啶橙 荧光淬灭进一步〜200秒,直到它稳定。 启动MdtM介导,质子驱动反运输通过加入底物 这种情况下胆汁钠胆酸钠)转化成倒置泡囊混合物。  我们加入12.5μl的250mM储备溶液在高纯度水中  以在〜2.0的比色杯中得到胆酸钠的最终浓度 mM。对于其他底物,我们建议测试范围从1 mM到  100mM以建立给出最佳脱猝灭的浓度 信号。加入底物后,应该有即时的 猝灭(上升)的吖啶橙荧光发射信号(见  图1a)由于所建立的ΔpH的耗散作为结果 MdtM介导的胆酸钠/H + +抗末端活性和伴随的 囊泡腔的碱化
    5. 记录荧光 脱猝信号50秒,使反向反应达到a 稳定状态(如通过荧光信号的平台所观察到的)。 加入质子载体CCCP至〜100μM的终浓度 (1.6μl的在乙醇中制备的100mM储备液) 应该执行以消除ΔpH驱动的,MdtM介导的反运输 活动。 加入CCCP将导致进一步的脱落 随着ΔpH消失,荧光信号(参见图1)。 记录 荧光信号进一步〜50秒,然后终止采集   并保存电子数据
    6. 将比色杯内容物倒入a 合适的废液容器,并用乙醇彻底清洗试管 然后高纯度水。 用压缩空气小心地干燥小杯。

代表数据


图1.当将胆汁盐加入到E的倒囊泡时,吖啶橙的荧光淬灭/去淬灭的代表性测量。过量产生重组(a)野生型MdtM或作为对照,(b)功能障碍的MdtM D22A突变体的大肠杆菌TO114细胞。通过加入DL-乳酸钠。如图所示,将胆酸钠加入到囊泡中以引发转运反应,并且将CCCP用于消散ΔpH。在添加胆酸钠时在对照实验(图b)中观察到的荧光猝灭是由于染色体编码的MdtM的反向端活性。以每秒计数(cps)测量荧光强度。请注意,您测量的荧光强度可能因仪器设置方式而异。上述迹线代表在至少两种单独的反转囊泡制备物上一式三份进行的实验的代表。

笔记

  1. 为了确保重现性,测定应至少进行两次单独的倒置泡囊制备,一式三份。
  2. 如果荧光信号不淬灭或增强,则囊泡不能保持完整性或不被倒转,并且其制备需要重复。
  3. 与依赖于荧光检测的所有测定一样,必须进行强有力的对照以确保任何检测到的转运活性可以明确地归因于感兴趣的蛋白质。在我们的实验中,我们 使用过表达MdtM D22A(MdtM的功能失调点突变体)的反向囊泡作为阴性对照(参见图1b)。
  4. 如果该方法用于检测金属离子/H sup/+反向运动活性,则使用从大肠杆菌的反转录物缺陷型TO114菌株产生的反转泡囊是重要的,因为存在于细菌中的至少四种其它转运蛋白(NhaA,NhaB,ChaA和MdfA)催化单价金属阳离子/H + 交换。
  5. 最后,如果该方案用于比较野生型和突变转运蛋白的反向运动活性,则必须定量(通常通过免疫检测方法)反向囊泡膜中存在的靶蛋白的量,以确保H + 吸收仅由于转运蛋白活性的差异,而不是表达水平的差异。

食谱

  1. LBK琼脂(100ml) 1.0克胰蛋白酶
    0.5g酵母提取物
    0.745克KCl
    1.5克琼脂 用高纯水补充至100ml,然后高压灭菌
    当溶液仍然是液体并温暖触摸时,加入100μg/ml羧苄青霉素进行选择 加入到无菌条件下的培养皿中
  2. LBK液体培养基(1 L) 10g胰蛋白胨
    5g酵母提取物
    7.45克KCl
    用高纯水补充至1000ml,然后高压灭菌
  3. Tris /胆碱/二硫苏糖醇/蔗糖(TCDS)缓冲液(1L) 由10mM Tris-HCl(pH7.5),140mM氯化胆碱,0.5mM二硫苏糖醇和250mM蔗糖组成。
    1M Tris-HCl(pH7.5)10ml
    2 M氯化胆碱70 ml
    1M蔗糖250ml
    用高纯度水补充至999.5毫升,然后检查pH值,如有必要用盐酸调整
    高压灭菌或无菌过滤并在4°C下保存
    在使用前立即从冷冻的1M储备液中加入0.5ml二硫苏糖醇
  4. 运输测定缓冲液(100 ml)
    包括10mM BisTris丙烷(pH 7.2),5mM MgSO 4,1μM吖啶橙
    100mM BisTris丙烷(pH7.2)10ml 2M MgSO 4 0.25ml
    10mM吖啶橙10μl
    用高纯水然后用无菌过滤器补充至100ml 吖啶橙是光敏感的,所以运输测定缓冲液和吖啶橙色股票应该存储在琥珀色瓶子或在避光在4℃在黑暗的容器
  5. 200mM DL-乳酸钠溶液(10ml) 0.45ml 50%DL-乳酸钠水溶液
    用高纯水
    补充至10ml 无菌过滤并在4℃下保存

致谢

    这项工作由BBSRC拨款BB/K014226/1(向CJL)支持。 以上描述的方案改变自之前报道的方案(Resch等人,2010)。

参考文献

  1. Paul,S.,Alegre,K.O.,Holdsworth,S.R.,Rice,M.,Brown,J.A.,McVeigh,P.,Kelly,S.M.and Law,C.J。(2014)。 主要促进者超家族的单组分多药转运蛋白是网络的一部分,可保护 Escherichia coli from bile salt stress。 Mol Microbiol 92(4):872-884。
  2. Reddy,V.S.,Shlykov,M.A.,Castillo,R.,Sun,E.I。和Saier,M.H.,Jr。(2012)。 主要促进者超家族(MFS)重新审核 FEBS J 279(11):2022-2035。
  3. Resch,C.T.,Winogrodzki,J.L.,Patterson,C.T.,Lind,E.J.,Quinn,M.J.,Dibrov,P.and Hase,C.C。(2010)。 推定的Na + /H + 霍乱弧菌的转运体,Vc-NhaP2,在体内介导特异性K + /H + 交换/a> Biochemistry 49(11):2520-2528。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Holdsworth, S. R. and Law, C. J. (2014). Measurement of Proton-driven Antiport in Escherichia coli. Bio-protocol 4(21): e1278. DOI: 10.21769/BioProtoc.1278.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。