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Heterologous Expression and Purification of the Magnesium Transporter A (MgtA) in Escherichia coli
在大肠杆菌中异源表达镁离子转运蛋白(MgtA)并纯化   

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Abstract

The magnesium transporter A (MgtA) is a magnesium transporting P-type ATPase present in prokaryotes and plants (Subramani et al., 2016). In Salmonella typhimurium and Escherichia coli (E. coli), MgtA is expressed only in magnesium limiting conditions and plays an important role in Mg2+ homeostasis (Groisman et al., 2013). The transcription of mgtA is regulated by the two-component system PhoP/PhoQ (Soncini et al., 1996; Kato et al., 1999). The membrane bound histidine kinase, PhoQ, senses low Mg2+ concentration in the periplasmic space and phosphorylates its cognate response regulator, PhoP, which initiates mgtA transcription (Groisman et al., 2013). MgtA is targeted to the plasma membrane and facilitate the bacterial survival under low Mg2+ condition, by importing Mg2+ into the cytoplasm. The MgtA homolog in petunia (PH1) is found in the vacuolar membrane and involved with the coloration of the flower petals (Faraco et al., 2014). As a first step towards understanding the molecular details of MgtA Mg2+ transport, we describe a detailed protocol for the purification of E. coli MgtA that can be used for biochemical and biophysical studies. Recombinant E. coli MgtA with hexa histidine tag at the N-terminus was cloned from E. coli DH5α and over expressed in the E. coli C43(DE3) by fermentation to an OD > 6. Cell lysis was performed in a high pressure homogenizer and the membranes were isolated by ultracentrifugation. Membrane proteins were solubilized with the detergent dodecyl-β-D maltoside. MgtA was purified by affinity and size exclusion chromatography. Final yields of purified MgtA reach ~1 mg MgtA per 3 g of wet cell pellet.

Background

Recently we have reported that the purified MgtA from E. coli is highly dependent on lipids for its function and studied the enzyme kinetics in vitro (Subramani et al., 2016). The protocol described here is the detailed description going through every single step of the purification that should yield monodisperse detergent solubilized MgtA, earlier described in Subramani et al. (2016). The protocol will in addition present notes that describe critical points and observations made in the process.

Materials and Reagents

  1. Disposable cup, 100 ml, PP (polypropylene) (SARSTEDT, catalog number: 75.562.105 )
    Note: It is used to store cell pellet.
  2. 70 ml polycarbonate ultracentrifugation tube
  3. C43(DE3) (Lucigen, catalog number: 60446 )
  4. PfuUltra II Fusion HS DNA polymerase (Agilent Technologies, catalog number: 600670 )
  5. LB broth powder (Sigma-Aldrich, catalog number: L3022 )
  6. Kanamycin (Sigma-Aldrich, catalog number: K1377 )
  7. Polypropylene glycol P2000 (Sigma-Aldrich, catalog number: 81380 )
  8. Gistex® LS Ferm - yeast extract (Fermia AB, Sweden)
  9. Glycerol (VWR, catalog number: 24388.295 )
  10. Isopropyl β-D-thiogalactopyranoside (IPTG) (Biosynth, catalog number: I-8000 )
  11. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, Catalog number: L4509 )
  12. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  13. Bromophenol blue (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP115-25 )
  14. Penta·His HRP Conjugate Kit (QIAGEN, catalog number: 34460 )
    Note: This is antibody against the His-tag and we used it for Western-blot analysis of 6x His-tagged MgtA. This antibody is conjugated to horseradish peroxidase (HRP), so there is no requirement for the use of secondary antibody.
  15. HEPES (Sigma-Aldrich, catalog number: H3375 )
  16. Potassium sulfate (K2SO4) (Sigma-Aldrich, catalog number: P9458 )
  17. Deoxyribonuclease I (Sigma-Aldrich, catalog number: DN25 )
  18. Phenylmethylsulfonyl fluoride (Sigma-Aldrich, catalog number: P7626 )
  19. Dodecyl-β-D maltoside (Chemical point, catalog number: CP69227-93-6-BULK )
  20. Imidazole (Sigma-Aldrich, catalog number: 56750 )
  21. Dithiothreitol (Biosynth, catalog number: D-8200 )
  22. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 221473 )
  23. Trizma® base (biotechnology performance certified) (Sigma-Aldrich, catalog number: T6066 )
  24. PageRulerTM prestained protein ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 26616 )
  25. LB broth (see Recipes)
  26. LB kanamycin plate (see Recipes)
  27. Growth media (see Recipes)
  28. 1x SDS-PAGE loading buffer (see Recipes)
  29. Buffer A (see Recipes)
  30. Buffer B (see Recipes)
  31. Buffer C (see Recipes)
  32. Buffer D (see Recipes)
  33. Buffer E (see Recipes)

Equipment

  1. Incubator
  2. Baffled bottom Erlenmeyer flask (500 ml) (Saveen & Werner, catalog number: 00125-54 )
    Note: These flasks have four baffles (flow directing ridges). It interrupts the circular flow of media and increases its gas exchange surface, thereby increasing the oxygenation of culture media when compared to a normal Erlenmeyer flask.
  3. Bottle with screw caps, used for fermentation (Saveen & Werner, catalog number: 88-2L )
  4. LEX (Large-scale Expression) 48 bioreactor system (Epiphyte Three, Harbinger Biotechnology and engineering, model: LEX-48 Bioreactor )
  5. High pressure homogenizer (used to lyse cells) (Avestin, model: EmulsiFlex C3 )
  6. Potter-Elvehjem-type tissue homogenizer (30 ml) (WHEATON, catalog number: 358049 )
  7. 70 ml polycarbonate ultracentrifugation tube assembly (Beckman Coulter, catalog number: 355622 )
  8. 45 Ti rotor (Beckman Coulter, catalog number: 339160 )
  9. OptimaTMXE (Beckman Coulter, catalog number: A99833 )
  10. Column HP Histrap, 5 ml (GE Healthcare, catalog number: 17-5248-02 )
  11. HiLoad 16/600 Superdex 200 pg column (GE Healthcare, catalog number: 28-9893-35 )
  12. Vivaspin 20 MWCO 50,00 PES (Saveen & Werner, catalog number: VS2032 )

Software

  1. Image LabTM software
  2. UNICORN software associated with ÄKTA purifier

Procedure

  1. Overexpression of MgtA
    The mgtA gene was amplified from E. coli DH5α genomic DNA using PfuUltra II Fusion HS DNA polymerase with the primers described earlier (Subramani et al., 2016). The reaction mixture and PCR conditions were made as suggested by the PfuUltra II Fusion HS DNA polymerase manual. The amplified gene was inserted between NcoI and XhoI restriction sites in pETM11 vector (EMBL). Thus the expressed MgtA will include 6x His tag followed by a tobacco etch virus (TEV) protease site at the N-terminus.
    1. Transform the pETM11(mgtA) plasmid into C43(DE3) competent cells and plate on LB kanamycin plate and incubate at 37 °C for 16 h.
    2. Select 5 colonies at random and inoculate in a 500 ml baffled bottom Erlenmeyer flask containing 200 ml LB media with 50 µg/ml of kanamycin. Incubate the flask at 37 °C with 150 rpm for 16 h.
    3. For large scale over expression of MgtA in C43(DE3) cells, we used the LEX 48 bioreactor system (Harbinger Technology LEXTM).
      Note: The LEX system delivers filtered compressed air through a sparger that passes through the screw cap. It provides both aeration and mixing of bacterial culture in the screw cap bottle. The culture flasks are placed in a temperature controlled water bath and the LEX 48 can accommodate 24 x 2 L standard screw cap bottles. Normally only 75% of the bottle capacity is used to avoid overflow of media during aeration.
    4. Inoculate 1% of overnight culture into 2 L screw cap bottles containing 1.5 L of growth media, 50 µg/ml of kanamycin and 1.5 ml of polypropylene glycol (PPG) P2000 (antifoaming agent).
    5. Place the bottles in the Lex 48 bioreactor water bath. Set the temperature of the water bath at 37 °C and initiate airflow through the spargers.
    6. Measure the optical density at 600 nm (OD600) every hour. The antifoaming agent PPG P2000 can become cloudy under these conditions and can be cleared upon incubation in ice for 5 min. Therefore, prior to the measurement at OD600, withdraw 1 ml of culture from the 2 L bottles and incubate on ice for 5 min.
    7. When the OD600 is ~0.6-2, stop the airflow and cool the water bath to 17 °C by adding ice. Leave the culture bottles at 17 °C for 30 min to ensure an equivalent temperature of the bacterial culture. In order to check the level overexpression by Western blot (see step 12), pellet 1 ml of bacterial culture at 12,000 x g, 4 °C and mark as SUI (uninduced).
    8. Add 1 mM of IPTG and an additional 1.5 ml of PPG P2000 to each bottle. Begin the airflow and leave the bottles at 17 °C for 16 h with continuous airflow.
    9. To terminate the induction, transfer the culture bottles to ice. Pellet 1 ml of bacterial culture from each bottle at 12,000 x g, 4 °C and mark as SI (induced) for Western blot analysis.
    10. Centrifuge rest of the bacterial culture at 7,000 x g for 20 min at 4 °C and divide the resulting cell pellet into 50 g aliquots in 100 ml disposable cups and store at -20 °C until further use.
    11. Suspend the SUI and SI sample pellets in 1x SDS-PAGE loading buffer to normalize the OD600 to 10 and incubate at RT for 5 min. Centrifuge the lysed cells at 20,000 x g for 20 min at 4 °C. For Western blot analysis use 10 µl of the supernatant and blot against Penta·His HRP conjugated antibody to check MgtA overexpression.
      Note: Confirmation of MgtA overexpression by Western blot against Penta·His HRP antibodies is highly recommended before beginning protein purification process as the detergents used during purification process are expensive and any problem associated with overexpression can be identified at this stage. Do not boil the samples used for SDS-PAGE and Western blot analysis.

  2. Cell lysis and solubilisation
    1. Thaw the frozen cell pellet on ice and suspend in buffer A at 1:10 (wt/vol) ratio.
    2. Lyse the cells using EmulsiFlex-C3, a high pressure homogenizer (HPH) by passing the cell suspension three times at 11,000 psi. Always store the samples on ice unless otherwise stated.
      Note: We did not test the other lysis methods like sonication and bead beater. But we believe, that they could also be used. The protein yield should be expected to vary depending on the lysis method.
    3. Centrifuge the cell lysate at 20,000 x g for 20 min at 4 °C to remove unlysed cells and inclusion bodies.
    4. Collect the supernatant and centrifuge at 100,000 x g for 2 h at 4 °C to pellet both the inner and outer membrane (mixed membranes).
      Note: This step requires ultracentrifuge with the rotors and tubes selected according to the volume of the sample. For example, if the sample size is 70 ml, then 45 Ti rotor and 70 ml polycarbonate ultracentrifugation tube can be used with ultracentrifuge OptimaTMXE. It is important to fill the tubes used for ultracentrifugation until the neck of the tube with possibly no gap between the liquid and cap of the tube to avoid the implosion of tubes during centrifugation.
    5. Suspend the resulting mixed membrane pellet in buffer B using Potter-Elvehjem-type tissue homogenizer with 10 strokes. Use 1:10 (wt/vol) mixed membrane to buffer B ratio. Before adding the required amount of buffer B to membrane pellet, remove 10 ml to dissolve the detergent.
      Note: The membrane suspension can be flash frozen with liquid nitrogen and stored at -80 °C if not used immediately.
    6. We used 1% β-dodecyl maltoside (β-DDM) to solubilize the membrane proteins. Weigh the required amount of detergent powder and dissolve in the reserved 10 ml of buffer B.
    7. Add the dissolved β-DDM drop wise into the mixed membrane suspension while stirring at 150 rpm, 4 °C. The suspension is left to stir for another 60 min at 4 °C.

  3. Purification of MgtA
    1. Prior to use, wash the Histrap HP 5 ml column with 5 column volume (CV) of degassed MQ water and equilibrate with 10 CV of buffer C.
    2. Add 20 mM imidazole (pH 7.6) to the sample prepared in step B7 and load on the Histrap column at 2 ml/min.
    3. After loading solubilized membrane sample, wash the Histrap column with 10 CV of buffer C and 3 CV of 15% buffer D at 3 ml/min. We observed a small peak (Figure 1A, peak 1) while washing with 15% buffer D.
    4. Elute MgtA from the Histrap column by passing 50% buffer D at 3 ml/min. We observed a relatively large peak (Figure 1A, peak 2) while washing with buffer D.
    5. Take 5 µl sample from each fraction and perform SDS-PAGE electrophoresis with 12% polyacrylamide gels.
    6. We observed that Peak 1 contained a small proportion of MgtA along with other impurities, whereas peak 2 contained > 80% pure MgtA, as assessed by SDS-PAGE (Figure 1A).
    7. Pool the fractions corresponding to peak 2 and concentrate to 6 mg/ml using a vivaspin 20 (protein concentrator with 50 kDa cutoff).
    8. While concentrating the fractions, prepare a HiLoad 16/600 Superdex 200 pg column connected to the ÄKTA purifier by washing with 1.5 CV of degassed MQ and 1.5 CV of buffer E at 0.5 ml/min.
    9. Load 1 ml of concentrated MgtA (6 mg/ml) on the HiLoad 16/600 Superdex 200 pg column at 0.5 ml/min and pass 1.5 CV of buffer E.
    10. We observed a small peak at the void volume (44 ml), followed by a major monodisperse peak (Figure 1B, peak S1) at ~55 ml.
    11. For SDS-PAGE analysis, load 5 µl of fractions corresponding to the observed peaks on 12% polyacrylamide gels. We observed that the void peak contained mostly impurities, whereas peak S1 contained MgtA at > 95% purity as analyzed by SDS-PAGE (Figure 1B).
    12. Pool the fractions from peak S1 and concentrate to 3 mg/ml using vivaspin 20.
    13. Divide the concentrated MgtA as 50 µl aliquots and flash freeze with liquid nitrogen and store at -80 °C.
    14. In our experience, the protein was stable under these conditions for more than 1 year.
       

      Figure 1. Representative figures of MgtA purification profile. A. Chromatogram of Histrap FF affinity column (left). The fractions from Peak 1 (lanes 1-4) and Peak 2 (lanes 6-11) were analyzed using SDS-PAGE and the Coomassie stained gel (right) shows the purity of the respective peaks. B. Chromatogram of size exclusion chromatography (left). The fractions from void (lanes 1-5) and Peak S1 (lanes 7-14) were analyzed using SDS-PAGE and the Coomassie stained gel (right) shows the purity of the respective peaks. In both SDS-PAGE gel pictures, MgtA runs at 100 kDa.

Data analysis

The data for chromatograms in Figure 1 were obtained from UNICORN software associated with ÄKTA purifier and the graphs were plotted using GraphPad Prism6. All the SDS-PAGE gels were scanned using Bio-Rad ChemiDoc XRS+ system and analyzed using Image LabTM software.

Recipes

  1. LB broth
    Mix 40 g of LB broth powder in 1 L of MQ and autoclave
  2. LB kanamycin plate
    Mix 40 g of LB broth powder and 7.5 g of LB agar and autoclave
    Prepare LB plate with 50 µg/ml kanamycin
  3. Growth media
    40 g of LB broth powder from Sigma-Aldrich (see Materials and Reagents)
    10 g of Fistex LS Ferm yeast extract
    Add 1 L of MQ and autoclave. Before inoculation, add 10 ml of 1 M Tris-Hcl pH 7.4 and 5 ml of glycerol
  4. 1x SDS-PAGE loading buffer
    2% SDS
    10% glycerol
    5% β-mercaptoethanol
    0.002% bromophenol blue
    63 mM Tris-HCl, pH 6.8
  5. Buffer A
    50 mM HEPES, pH 7.0 (KOH)
    100 mM K2SO4
    10% glycerol
    1 mM PMSF
    5 mM β-mercaptoethanol
    1 μg/ml DNase
  6. Buffer B
    25 mM HEPES, pH 7.0 (KOH)
    100 mM K2SO4
    5% glycerol
    1 mM PMSF
    5 mM β-mercaptoethanol
  7. Buffer C
    25 mM HEPE, pH 7.0 (KOH)
    100 mM K2SO4
    5% glycerol
    1 mM PMSF
    5 mM β-mercaptoethanol
    20 mM Imidazole, pH 7.6
    3 CMC β-DDM
  8. Buffer D
    25 mM HEPES, pH 7.0 (KOH)
    100 mM K2SO4
    5% glycerol
    1 mM PMSF
    5 mM β-mercaptoethanol
    300 mM Imidazole pH 7.6
    3 CMC β-DDM
  9. Buffer E
    25 mM HEPES, pH 7.0 (KOH)
    100 mM K2SO4
    5% glycerol
    1 mM dithiothreitol
    3 CMC β-DDM

Note: The buffers used for chromatography were always filtered using the NalgeneTM reusable bottle top filter connected to vacuum pump. Buffers were degassed with the same set up, but by closing the filter chamber while the vacuum still on and with constant stirring. Buffers were degassed until no bubbles were observed in the solution.

Acknowledgments

The Norwegian Research Council Funding (F-RIMEDBIO) #ES486454 and NCMM core Funding supported this study.

References

  1. Faraco, M., Spelt, C., Bliek, M., Verweij, W., Hoshino, A., Espen, L., Prinsi, B., Jaarsma, R., Tarhan, E., de Boer, A. H., Di Sansebastiano, G. P., Koes, R. and Quattrocchio, F. M. (2014). Hyperacidification of vacuoles by the combined action of two different P-ATPases in the tonoplast determines flower color. Cell Rep 6(1): 32-43.
  2. Groisman, E. A., Hollands, K., Kriner, M. A., Lee, E. J., Park, S. Y. and Pontes, M. H. (2013). Bacterial Mg2+ homeostasis, transport, and virulence. Annu Rev Genet 47: 625-646.
  3. Kato, A., Tanabe, H. and Utsumi, R. (1999). Molecular characterization of the PhoP-PhoQ two-component system in Escherichia coli K-12: identification of extracellular Mg2+-responsive promoters. J Bacteriol 181(17): 5516-5520.
  4. Soncini, F. C., Garcia Vescovi, E., Solomon, F. and Groisman, E. A. (1996). Molecular basis of the magnesium deprivation response in Salmonella typhimurium: identification of PhoP-regulated genes. J Bacteriol 178(17): 5092-5099.
  5. Subramani, S., Perdreau-Dahl, H. and Morth, J. P. (2016). The magnesium transporter A is activated by cardiolipin and is highly sensitive to free magnesium in vitro. Elife 5.

简介

镁转运蛋白A(MgtA)是存在于原核生物和植物中的镁转运P型ATP酶(Subramani等,2016)。在鼠伤寒沙门氏菌和大肠杆菌(Escherichia coli)(大肠杆菌)中,MgtA仅在镁限制条件下表达,并在Mg2 +稳态中起重要作用(Groisman等,2013)。 mgtA的转录由双组分系统PhoP / PhoQ(Soncini等人,1996; Kato等,1999)调节。膜结合的组氨酸激酶PhoQ感测周质空间中的低Mg2 +浓度,并磷酸化其启动mgtA转录的同源反应调节因子PhoP(Groisman等,2013)。通过将Mg2 +导入细胞质,MgtA靶向质膜并促进低Mg2 +条件下的细菌存活。矮牵牛的MgtA同源物(PH1)在液泡膜中发现,涉及花瓣的着色(Faraco等,2014)。作为理解MgtA Mg2 +转运的分子细节的第一步,我们描述了可用于生物化学和生物物理学研究的大肠杆菌MgtA的纯化的详细方案。在大肠杆菌DH5α中克隆了在N末端具有六氨基组氨酸标签的重组大肠杆菌MgtA,并在大肠杆菌C43(DE3)中通过发酵至OD> 6进行表达。细胞裂解在高压均化器并通过超速离心分离膜。用洗涤剂十二烷基-β-D麦芽糖苷溶解膜蛋白质。通过亲和力和尺寸排阻色谱纯化MgtA。纯化的MgtA的最终产量每3g湿细胞沉淀达到〜1mg MgtA。

材料和试剂

  1. 一次性杯,100ml,PP(聚丙烯)(SARSTEDT,目录号:75.562.105)
    注意:它用于储存细胞沉淀。
  2. 70ml聚碳酸酯超速离心管
  3. C43(DE3)(Lucigen,目录号:60446)
  4. PfuUltra II Fusion HS DNA聚合酶(Agilent Technologies,目录号:600670)
  5. LB肉汤粉末(Sigma-Aldrich,目录号:L3022)
  6. 卡那霉素(Sigma-Aldrich,目录号:K1377)
  7. 聚丙二醇P2000(Sigma-Aldrich,目录号:81380)
  8. Gistex LS Ferm酵母提取物(Fermia AB,Sweden)
  9. 甘油(VWR,目录号:24388.295)
  10. 异丙基β-D-硫代吡喃半乳糖苷(IPTG)(Biosynth,目录号:I-8000)
  11. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L4509)
  12. β-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  13. 溴酚蓝(Thermo Fisher Scientific,Fisher scientific,目录号:BP115-25)
  14. Penta·His HRP缀合试剂盒(QIAGEN,目录号:34460)
    注意:这是针对His标签的抗体,我们将其用于6x His标记的MgtA的Western印迹分析。该抗体与辣根过氧化物酶(HRP)结合,因此不需要使用二抗。
  15. HEPES(Sigma-Aldrich,目录号:H3375)
  16. 硫酸钾(K 2 SO 4)(Sigma-Aldrich,目录号:P9458)
  17. 脱氧核糖核酸酶I(Sigma-Aldrich,目录号:DN25)
  18. 苯甲基磺酰氟(Sigma-Aldrich,目录号:P7626)
  19. 十二烷基-β-D麦芽糖苷(化学点,目录号:CP69227-93-6-BULK)
  20. 咪唑(Sigma-Aldrich,目录号:56750)
  21. 二硫苏糖醇(Biosynth,目录号:D-8200)
  22. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:221473)
  23. (生物技术性能认证)(Sigma-Aldrich,目录号:T6066)
  24. PageRuler TM 预染蛋白梯(Thermo Fisher scientific,Thermo scientific TM ,目录号:26616)
  25. LB肉汤(见配方)
  26. LB卡那霉素板(见配方)
  27. 生长培养基(参见食谱)
  28. 1x SDS-PAGE上样缓冲液(见配方)
  29. 缓冲区A(参见配方)
  30. 缓冲液B(参见配方)
  31. 缓冲区C(参见配方)
  32. 缓冲区D(参见配方)
  33. 缓冲区E(参见配方)

设备

  1. 孵化器
  2. 带挡板的锥形瓶(500ml)(Saveen& Werner,目录号:00125-54)
    注意:这些烧瓶有四个挡板(流动引导脊)。它阻断了介质的循环流动,增加了其气体交换表面,从而与正常的锥形瓶相比,增加了培养基的氧合作用。
  3. 用于发酵的带螺旋盖的瓶(Saveen& Werner,目录号:88-2L)
  4. LEX(大规模表达)48生物反应器系统(Epiphyte Three,Harbinger Biotechnology and engineering,model:LEX-48 Bioreactor)
  5. 高压匀浆器(用于裂解细胞)(Avestin,型号:EmulsiFlex C3)
  6. Potter-Elvehjem型组织匀浆器(30ml)(WHEATON,目录号:358049)
  7. 70ml聚碳酸酯超速离心管组件(Beckman Coulter,目录号:355622)
  8. 45Ti转子(Beckman Coulter,目录号:339160)
  9. Optima TM XE(Beckman Coulter,目录号:A99833)
  10. 柱HP Histrap,5ml(GE Healthcare,目录号:17-5248-02)
  11. HiLoad 16/600 Superdex 200pg柱(GE Healthcare,目录号:28-9893-35)
  12. Vivaspin 20 MWCO 50,00 PES(Saveen& Werner,目录号:VS2032)

软件

  1. Image Lab TM 软件
  2. 与ÄKTA净化器关联的UNICORN软件

程序

  1. MgtA的过表达
    从em扩增了mgtA 基因。 (Subramani等人,2016),使用PfuUltra II Fusion HS DNA聚合酶与大肠杆菌DH5α基因组DNA杂交。如PfuUltra II Fusion HS DNA聚合酶手册所建议的,制备反应混合物和PCR条件。将扩增的基因插入pETM11载体(EMBL)中的NcoI和XhoI 限制性位点之间。因此,表达的MgtA将包括6x His标签,随后在N端具有烟草蚀纹病毒(TEV)蛋白酶位点。
    1. 将pETM11(mgtA)质粒转化到C43(DE3)感受态细胞中,并在LB卡那霉素平板上平板,并在37℃孵育16小时。
    2. 随机选择5个菌落并接种在含有200ml含有50μg/ml卡那霉素的LB培养基的500ml带挡板的锥形瓶中。将烧瓶在37℃下以150rpm孵育16小时
    3. 对于MgtA在C43(DE3)细胞中的大规模过表达,我们使用LEX 48生物反应器系统(Harbinger Technology LEX TM )。
      注意:LEX系统通过穿过螺旋盖的分布器提供过滤的压缩空气。它提供在螺旋盖瓶中的细菌培养物的曝气和混合。将培养瓶置于温控水浴中,LEX 48可容纳24×2L标准螺旋盖瓶。通常只有75%的瓶容量用于避免曝气期间介质溢出。
    4. 将1%的过夜培养物接种到含有1.5L生长培养基,50μg/ml卡那霉素和1.5ml聚丙二醇(PPG)P2000(消泡剂)的2L螺旋盖瓶中。
    5. 将瓶子放在Lex 48生物反应器水浴中。将水浴的温度设置在37°C,启动气流通过喷头。
    6. 每小时测量600nm的光密度(OD 600)。消泡剂PPG P2000在这些条件下可以变混浊并且可以在冰中孵育5分钟时澄清。因此,在OD 600测量之前,从2L瓶中取出1ml培养物并在冰上孵育5分钟。
    7. 当OD <600>为〜0.6-2时,停止气流,并通过加入冰将水浴冷却至17℃。离开培养瓶在17℃下30分钟,以确保细菌培养物的当量温度。为了通过Western印迹检查水平过表达(参见步骤12),在12,000xg,4℃下沉淀1ml细菌培养物,并标记为S sub UI(未诱导的)。
    8. 向每个瓶中加入1mM IPTG和另外1.5ml PPG P2000。开始气流,并将瓶子在17°C下保持16小时,持续气流
    9. 要终止诱导,将培养瓶转移到冰上。沉淀1ml来自每个瓶的12,000×g,4℃的细菌培养物,标记为S I(诱导)用于蛋白质印迹分析。
    10. 在4℃下以7000xg离心细菌培养物的剩余部分20分钟,并将所得细胞沉淀物分成在100毫升一次性杯中的50克等分试样,并储存在-20℃直至进一步使用。 br />
    11. 在1×SDS-PAGE上样缓冲液中悬浮S Sub UI和S I样品沉淀物以将OD 600标准化为10,并在室温下孵育5小时min。在4℃下将裂解的细胞以20,000×g离心20分钟。对于蛋白质印迹分析,使用10μl上清液和针对Penta·His HRP偶联抗体的印迹以检查MgtA过表达。
      注意:在开始蛋白质纯化过程之前强烈建议通过蛋白质印迹对Penta·His HRP抗体的MgtA过表达的证实,因为在纯化过程中使用的去污剂是昂贵的,并且在该阶段可以鉴定与过表达相关的任何问题。不要煮沸用于SDS-PAGE和Western印迹分析的样品
  2. 细胞裂解和溶解
    1. 在冰上解冻冷冻的细胞沉淀,并以1:10(wt/vol)比率悬浮在缓冲液A中
    2. 使用EmulsiFlex-C3,高压匀浆器(HPH),通过使细胞悬浮液在11,000psi下通过三次来裂解细胞。除非另有说明,否则始终将样品储存在冰上。
      注意:我们没有测试其他裂解方法,如超声处理和珠磨机。但我们相信,他们也可以使用。蛋白质产量应该根据裂解方法而变化。
    3. 在4℃下以20,000×g离心细胞裂解物20分钟,以除去未裂解的细胞和包涵体。
    4. 收集上清液并在4℃下以100,000×g离心2小时以沉淀内膜和外膜(混合膜)。
      注意:此步骤需要超速离心机,根据样品的体积选择转子和管。例如,如果样品大小为70ml,那么45Ti转子和70ml聚碳酸酯超速离心管可以与超速离心机Optima TM sup/XE一起使用。重要的是将用于超速离心的管填充到管的颈部,在管的液体和盖之间可能没有间隙,以避免在离心期间管的内爆。
    5. 使用Potter-Elvehjem型组织匀浆器以10次冲程将所得混合膜沉淀物在缓冲液B中悬浮。使用1:10(wt/vol)混合膜与缓冲液B比。在向膜沉淀物中加入所需量的缓冲液B之前,取出10ml以溶解洗涤剂 注意:如果不立即使用,可以用液氮快速冷冻膜悬浮液并储存在-80℃。
    6. 我们使用1%β-十二烷基麦芽糖苷(β-DDM)来溶解膜蛋白。称量所需量的洗涤剂粉末,并溶解在保留的10ml缓冲液B中
    7. 将溶解的β-DDM滴加到混合的膜悬浮液中,同时在150rpm,4℃下搅拌。将悬浮液在4℃下再搅拌60分钟
  3. MgtA的纯化
    1. 使用前,用5柱体积(CV)的脱气MQ水洗涤Histrap HP 5 ml柱,并用10CV缓冲液C平衡。
    2. 向步骤B7中制备的样品中加入20mM咪唑(pH 7.6),并以2ml/min加载到Histrap柱上。
    3. 加载溶解的膜样品后,用10CV的缓冲液C和3CV的15%缓冲液D以3ml/min洗涤Histrap柱。我们观察到一个小峰(图1A,峰1),同时用15%缓冲液D洗涤
    4. 通过以3ml/min通过50%缓冲液D从Histrap柱洗脱MgtA。当用缓冲液D洗涤时,我们观察到相对较大的峰(图1A,峰2)
    5. 从每个部分取5μl样品,并用12%聚丙烯酰胺凝胶进行SDS-PAGE电泳
    6. 我们观察到峰1含有小比例的MgtA以及其它杂质,而峰2含有> 80%纯的MgtA,如通过SDS-PAGE评估的(图1A)
    7. 汇集对应于峰2的级分,并使用vivaspin 20(具有50kDa截留的蛋白质浓缩器)浓缩至6mg/ml。
    8. 在浓缩馏分时,通过用1.5CV脱气的MQ和1.5CV的缓冲液E以0.5ml/min洗涤来制备连接到ÄKTA净化器的HiLoad 16/600 Superdex 200pg柱。
    9. 在HiLoad 16/600 Superdex 200 pg柱上以0.5ml/min加载1ml浓缩的MgtA(6mg/ml),并通过1.5CV的缓冲液E.
    10. 我们在空隙体积(44ml)处观察到小峰,随后在约55ml处观察到主要的单分散峰(图1B,峰S1)。
    11. 对于SDS-PAGE分析,加载5μl对应于在12%聚丙烯酰胺凝胶上观察到的峰的级分。我们观察到空隙峰主要含有杂质,而峰S1含有MgtA> 95%的纯度,通过SDS-PAGE分析(图1B)
    12. 从峰S1中收集级分,并用维生素A 20浓缩至3mg/ml
    13. 将浓缩的MgtA分成50μl等分试样,并用液氮快速冷冻,并储存在-80℃。
    14. 根据我们的经验,蛋白质在这些条件下稳定超过1年  

      图1. MgtA纯化图谱的代表性数字。 A. Histrap FF亲和柱的色谱图(左)。使用SDS-PAGE分析峰1(泳道1-4)和峰2(泳道6-11)的级分,考马斯染色的凝胶(右)显示各峰的纯度。 B.尺寸排阻色谱的色谱图(左)。使用SDS-PAGE分析来自空白(泳道1-5)和峰S1(泳道7-14)的级分,考马斯染色的凝胶(右)显示各个峰的纯度。在两个SDS-PAGE凝胶图片中,MgtA运行在100kDa。

数据分析

图1中的色谱图的数据从与ÄKTA净化器相关的UNICORN软件获得,并且使用GraphPad Prism6对图进行绘图。使用Bio-Rad ChemiDoc XRS +系统扫描所有SDS-PAGE凝胶,并使用Image Lab TM supec软件分析。

食谱

  1. LB肉汤
    将40g LB肉汤粉末在1L MQ和高压釜中混合
  2. LB卡那霉素板
    混合40g LB肉汤粉末和7.5g LB琼脂和高压釜
    用50μg/ml卡那霉素制备LB平板
  3. 生长介质
    40g来自Sigma-Aldrich的LB肉汤粉末(见材料和试剂)
    10g Fistex LS Ferm酵母提取物
    加入1升MQ和高压釜。接种前,加入10ml的1M Tris-HCl pH7.4和5ml的丙三醇
  4. 1x SDS-PAGE上样缓冲液
    2%SDS
    10%甘油 5%β-巯基乙醇 0.002%溴酚蓝
    63 mM Tris-HCl,pH 6.8
  5. 缓冲区A
    50mM HEPES,pH 7.0(KOH)
    100mM K 2 SO 4
    10%甘油
    1mM PMSF
    5mMβ-巯基乙醇 1μg/ml DNase
  6. 缓冲区B
    25mM HEPES,pH 7.0(KOH)
    100mM K 2 SO 4 4/v/v 5%甘油 1mM PMSF
    5mMβ-巯基乙醇
  7. 缓冲区C
    25mM HEPE,pH 7.0(KOH)
    100mM K 2 SO 4 4/v/v 5%甘油 1mM PMSF
    5mMβ-巯基乙醇 20mM咪唑,pH7.6
    3 CMCβ-DDM
  8. 缓冲区
    25mM HEPES,pH 7.0(KOH)
    100mM K 2 SO 4 4/v/v 5%甘油 1mM PMSF
    5mMβ-巯基乙醇 300mM咪唑pH7.6
    3 CMCβ-DDM
  9. 缓冲区E
    25mM HEPES,pH 7.0(KOH)
    100mM K 2 SO 4 4/v/v 5%甘油 1mM二硫苏糖醇 3 CMCβ-DDM

注意:用于色谱的缓冲液总是使用连接到真空泵的Nalgene TM可重复使用的瓶顶过滤器过滤。用同样的设置使缓冲液脱气,但是通过关闭过滤室,同时真空仍然开启并且在不断搅拌下。将缓冲液脱气,直到在溶液中没有观察到气泡

致谢

挪威研究委员会资助(F-RIMEDBIO)#ES486454和NCMM核心资助支持这项研究。

参考文献

  1. Farerman,M.,Spelled,C.,Bliek,M.,Verweij,W.,Hoshino,A.,Espen,L.,Prinsi,B.,Jaarsma,R.,Tarhan, Di Sansebastiano,GP,Koes,R。和Quattrocchio,FM(2014)。  通过在叶绿体中两种不同的P-ATP酶的组合作用,液泡的超酸化确定花色。细胞报告6(1):32-43。 >
  2. Groisman,EA,Hollands,K.,Kriner,MA,Lee,EJ,Park,SYand Pontes,MH(2013)。  细菌Mg 2 + 稳态,转运和毒力。 Annu Rev Ge net 47:625-646。
  3. Kato,A.,Tanabe,H。和Utsumi,R。(1999)。  J Bacteriol 181(17):5516-5520。
  4. Soncini,FC,Garcia Vescovi,E.,Solomon,F.and Groisman,EA(1996)。  鼠伤寒沙门氏菌中镁剥夺反应的分子基础:鉴定PhoP调节基因。


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  5. Subramani,S.,Perdreau-Dahl,H. and Morth,JP(2016)。 
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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 Subramani and Morth. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Subramani, S. and Morth, J. P. (2016). Heterologous Expression and Purification of the Magnesium Transporter A (MgtA) in Escherichia coli. Bio-protocol 6(22): e2001. DOI: 10.21769/BioProtoc.2001.
  2. Subramani, S., Perdreau-Dahl, H. and Morth, J. P. (2016). The magnesium transporter A is activated by cardiolipin and is highly sensitive to free magnesium in vitro. Elife 5.e11407.
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