搜索

Analysis of Mycobacterial Protein Secretion
分枝杆菌蛋白质分泌物的分析   

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

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. Analysis of proteins secreted by Mtb has been of interest to the field of tuberculosis research since certain secreted proteins interact with the host to promote virulence, while others may be important antigens or serve as biomarkers of infection. Here, we describe a protocol to prepare whole cell extracts (WCE) and short term culture filtrate (CF) from Mtb or the vaccine strain Mycobacterium bovis- bacillus Calmatte- Guérin (BCG) (Mehra et al., 2013). These are both slow growing mycobacteria, but the same basic procedure can easily be adapted to analyze secreted proteins from rapidly growing mycobacteria, such as Mycobacterium smegmatis (Msmeg), a non-pathogenic species commonly used in the laboratory. The fractions obtained can be analyzed by western blotting to examine proteins of interest or by mass spectrometry if antibodies are not available or to examine the entire secretome. Genetic knockout mutants for the gene of interest serve as a negative control. Additionally, levels of a cytosolic protein such as the chaperone GroEL or the pyruvate dehydrogenase E2 component sucB (Rv2215/dlaT) should be assessed in the CF fraction to rule out the possibility that a positive signal in CF is due to bacterial lysis (see Figure 1). By varying the growth conditions of the strain, this in vitro secretion assay can be used to examine conditions that alter the secretome. We are thankful to Magnus Stiegedal for helpful tips on TCA (trichloroacetic acid) precipitation.

Keywords: Mycobacteria culture(结核分枝杆菌培养), Culture filtrates(培养滤液), Whole cell extracts(全细胞提取物), TCA precipitation(TCA沉淀), Bead beating(珠跳动)



Figure 1. Western analysis of secretion of EsxH by BCG. BCG containing an empty vector control and EsxG-EsxH-FLAG expression construct (FLAG tag at C terminal of EsxH) were analyzed for presence EsxH by anti-FLAG western in WCE and CF prepared as described in the protocol. DlaT was used as a loading control to indicate the degree of bacterial lysis.

Materials and Reagents

Note: All work with live Mtb must be performed in a Biosafety Level 3 (BSL3) facility.

  1. Middlebrook 7H9 Broth (Difco, catalog number: 271310 )
  2. Tween-80 (Sigma-Aldrich, catalog number: P4780 )
  3. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  4. Albumin-dextrose-catalase (ADC) (BD, catalog number: 212352 )
  5. Oleic-albumin-dextrose-catalase (OADC) (BD, catalog number: 212351 )
  6. Potassium phosphate (monobasic) (KH2PO4) (Sigma-Aldrich, catalog number: P9791 )
  7. L-asparagine monohydrate (Sigma-Aldrich, catalog number: A8381 )
  8. Citric acid monohydrate (Sigma-Aldrich catalog number: C1909 )
  9. Ferric ammonium citrate (Sigma-Aldrich catalog number: F5879 )
  10. Zinc sulfate monohydrate (ZnSO4.H2O) (Sigma-Aldrich, catalog number: 96495 )
  11. Magnesium sulfate heptahydrate (MgSO4.7H2O) (Sigma-Aldrich, catalog number: 230391 )
  12. Chelex 100 resin (Bio-Rad Laboratories, catalog number: 142-2822 )
  13. 100x Halt Protease Inhibitor single use cocktail (Pierce, catalog number: 1860932 )
  14. 100% trichloroacetic acid (TCA) (pre-chilled prior to use) (Sigma-Aldrich, catalog number: 49010 )
  15. Acetone (pre-chilled prior to use) (Sigma-Aldrich, catalog number: 32201 )
  16. Phosphate buffered saline (PBS) (Life Technologies, Gibco®, catalog number: 10010-023 )
  17. Bromophenol Blue, sodium salt (US Biological, catalog number: 12370 )
  18. Tris (MP Biomedicals, catalog number: 02194855 )
  19. Sodium Dodecyl Sulfate (SDS) (US Biological, catalog number: 18220 )
  20. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
  21. β-mercaptoethanol (2-ME) (Sigma-Aldrich, catalog number: M6250 )
  22. 7H9 complete media (see Recipes)
  23. Sauton’s media (see Recipes)
  24. Chelated sauton’s media (an alternative minimal media for mycobacterial growth) (see Recipes)
  25. Protein extraction buffer (see Recipes)
  26. 5x SDS-PAGE sample buffer (see Recipes)

Equipment

  1. Autoclave
  2. Steriflip-GV filter units (0.22 µM pore size) (Millipore, catalog number: SE1M179M6 )
  3. 20 ml syringes (BD, catalog number: 302830 )
  4. 0. 22 µM syringe filter units (33 mm) (Millipore, catalog number: SLGV033RS )
  5. Disposable Sterile Filter system (1L, 0.22 µm pore size) (Corning, catalog number: 09761104 )
  6. 0.1 mm zirconia/silica beads (Bio Spec Products, catalog number: 11079101z )
  7. 30 ml square media bottles (Nalgene®, catalog number: NE/2019-0030 )
  8. 125 ml square media  bottles (Nalgene®, catalog number: NE/2019-0125 )
  9. 50 ml falcon tubes (Corning, catalog number 430290 )
  10. 15 ml falcon tubes (Corning, catalog number: 430052 ) with plug seal caps
    Note: These 50 ml tubes are compatible with organic solvents and high speed centrifugation. Falcon tubes with these features can be used from different vendors.
  11. Microtubes (2 ml screw cap with O rings) (SARSTEDT AG, catalog number: 72.693 )
  12. Spectrophotometer
  13. Centrifuge with swinging bucket rotor for spinning down bacterial cultures (for example, Beckman Coulter, model: Allegra X-15R ; bench top centrifuge with SX4750 rotor)
    Notes:
    1. Msmeg and BCG should be handled according to institutional standards of practice for biosafety.
    2. Mtb cultures should be handled in biosafety level 3 facilities according to institutional standards of practice.
    3. Centrifuging BCG and Mtb requires appropriate aerosol containment.
  14. Beckman Aerosolve® canisters to contain aerosols during centrifugation of mycobacterial cultures (e.g. Beckman Coulter, catalog number: BK359232 )
  15. 37 °C shaking incubator
  16. Aerosol containment units for shaking BCG and Mtb liquid cultures in the shaking incubator
    Note: Incubator should be placed in BSL3 facility for Mtb cultures.
  17. High speed centrifuge for 50 ml polypropylene falcons used for TCA precipitation of CF (e.g. Beckman Coulter centrifuge with JLA16.2 rotor)
  18. 50 ml falcon adaptors for rotor JLA16.2
  19. Bead beater (Bio Spec Products, model: Minibead beater 16; http://www.biospec.com/product/34/mini_beadbeater/)
  20. Standard table top centrifuge with refrigeration
  21. Heating block for Eppendorf tubes (set to 95 °C)
  22. pH meter

Acronyms

  1. Mtb: Mycobacterium tuberculosis
  2. BCG: Mycobacterium bovis bacillus Calmatte- Guérin
  3. Msmeg: Mycobacterium smegmatis
  4. WCE: Whole cell extract
  5. CF: Culture filtrate
  6. DlaT: Rv2215/pyruvate dehydrogenase E2 component sucB protein of mycobacteria
  7. TCA: Trichloroacetic acid
  8. BSC: Biosafety cabinet
  9. OD600: Absorbance or Optical Density at wavelength of 600 nm

Procedure

  1. Inoculation and growth of mycobacterial cultures
    1.  Inoculate liquid cultures of mycobacteria from frozen stocks, prepared from mid-log phase mycobacterial cultures (OD600 between 0.5 to 1.0) that were frozen in 18% glycerol at -80 °C.
      1. For inoculation, the doubling rate of the mycobacterial species needs to be kept in mind. Msmeg doubles approximately every 3 h, whereas BCG and Mtb double approximately every 20 h in 7H9 complete media at 37 °C.
      2. The cultures are inoculated in 10 ml of 7H9 complete media in 30 ml square media bottles. Include appropriate antibiotics if required e.g. antibiotics can be included at appropriate concentration for selecting a plasmid when growing a transformed mycobacterial strain.
      3. The mycobacterial cultures are incubated at 37 °C with slow shaking at a speed of 90 rpm. BCG and Mtb cultures are placed in aerosol containment in a shaking incubator.
      4. For Msmeg, inoculate 200 µl from glycerol stock and grow overnight to saturation. Dilute the over-night culture 1:40 into fresh 7H9 complete media and grow to mid-log phase. For BCG and Mtb, inoculate 0.5 ml from frozen stock. It will take several days (~4 days) for the culture to reach mid-log phase and the exact time depends upon the initial inoculum density and growth conditions. 
    2. Measure the OD600 of the culture. Determine the culture volume required to give a final starting OD600 of at least 0.25 when resuspended in 25 ml of Sauton’s media [culture volume in ml = 25 ml x desired OD600 (e.g. 0.25)/OD600 of the experimental culture].
    3. Transfer this volume of culture media to a 15 ml falcon tube and pellet the bacterial cells by centrifuging at 1500 x g for 7 min at room temperature. Remove the supernatant and resuspend the pellet in PBS.  Again centrifuge at 1,500 x g for 7 min. This wash step is important to remove albumin carried over from 7H9 complete media.  
      Notes:  
      1. As per our institutional standards of practice for centrifuging Mtb and BCG cultures, falcon tubes are placed in centrifuge adaptors that are put in Aerosolve® canisters (mentioned in Equipment, item 14) inside class II biosafety cabinet (BSC II).
      2. These are removed from the hood and loaded into the rotor and centrifuged.
      3. After spin, the whole Aerosolve® canister with the falcon tubes is carried back to the BSC II hood and opened.
      4. This procedure contains aerosols produced during centrifugation. Mtb cultures should be handled in BSL3 facility.
    4. Remove the supernatant and resuspend the bacterial pellet in 1 ml of Sauton’s media. Transfer this to 125 ml square media bottle containing 24 ml of Sauton’s media (include appropriate antibiotics in Sauton’s media as required). Incubate in the shaking incubator at 37 °C.
       Notes:
      1. Sauton’s media is a minimal media in which mycobacteria grow more slowly than they do in 7H9 complete media.
      2. Alternatively, if the effect of metal ions on secretion is to be studied, one can use chelated Sauton’s media as the minimal media and add defined concentrations of metal ions.
    5. Monitor OD600 of the culture. Grow the culture to between 0.5 to 0.8 OD600. It takes about 6 h for Msmeg and 48 h for BCG and Mtb This procedure could be modified to inoculate the bacteria at a lower density and allow them to grow for a longer period of time, depending upon the goals of the experiment.

  2. Harvesting the bacterial culture and preparation of the WCE
    1. After incubating in Sauton’s media as described above, determine the OD600.
    2. Calculate the volume corresponding to 5 OD600 units. For example, if the OD600 of the culture is 0.5, then 10 ml will correspond to 5 OD units (desired volume = 5/OD600 of the experimental culture).
    3. Transfer 5 OD600 of the culture into a 15 ml falcon tube (to be used to prepare the WCE). Transfer the remaining culture into a 50 ml falcon tube (to be used to precipitate proteins from the CF). Centrifuge both tubes at 2,000 x g for 5 min at 4 °C.
    4. Pool the supernatant from both the 15 and 50 ml falcons into one unused 50 ml falcon tube and store on ice until you are ready to process it further (see below, preparation of CF in step C).  
    5. Wash the pellet in the 15 ml falcon with 5 ml of PBS by resuspending and centrifuging at 2,000 x g, 5 min, 4 °C.
    6. Discard the supernatant and resuspend the pellet again in 1 ml of PBS and transfer it to a 2 ml Sarstedt tube with O ring in the screw cap (for containing aerosols during bead beating).
    7. Spin on a table top centrifuge at 3,000 x g, 5min and discard the supernatant. To the pellet add 300 µl of protein extraction buffer with protease inhibitors. Resuspend the pellet by pippetting.
    8. Dispense 100 µl of 0.1 mm zirconia/silica beads into a 1.7 ml eppendorf and add this to the Sarstedt tube containing the pellet.
    9. Using a bead beater contained within a class II bio-safety cabinet (please read the bead beater manual for the operating procedure and also see the link provided in Equipment, item 19), bead beat the sample for 1 min at 3,450 oscillations/min and place it on ice for 2 min to cool. Repeat this twice. This sample is the WCE. Centrifuge the lysate at full speed for 2 min in a microcentrifuge to settle the froth before opening the tube.
      Notes:
      1. Alternate bead beaters may have different speeds and hence the user should optimize bead beating (speed and time) for maximal lysis of mycobacterial pellet.
      2. The degree of lysis can be established by plating the bacteria before and after bead beating for colony forming units.
    10. For SDS-PAGE, add 60 µl of 5x SDS-PAGE sample buffer to the Sarstedt tube and then gently mix by inversion. Incubate at 95 °C for 5 min. The heating step helps to denature the sample and also sterilize mycobacterial lysate after bead beating. The mycobacterial WCE can be stored at -80 °C.
      Note: The WCE of Mtb can generally be transferred out of BSL3 facility at this step as long as that is consistent with the institutional standards of practice.
    11. Before use, thaw at room temperature and then heat at 95 °C for 5 min. 20 µl of the WCE is often sufficient for SDS-PAGE and western analysis, although this amount will depend upon the target protein levels and the sensitivity of the detection method.

  3. TCA precipitation of the CF
    1. Filter the culture supernatant from step B9 through 0.22 µM filters using a 25 ml syringe into a fresh 50 ml falcon tube.
      Notes:
      1. Alternatively, it can be filtered by vacuum using 50 ml Steriflip-GV filter units (0.22 µM) to remove any bacteria from the supernatants.
      2. This can be done twice depending on the turbidity of the supernatant.
      3. Double filtration may also be required depending upon the standard operating procedures of the facility to transfer the CF from the BSL3 facility.
    2. Pre-chill trichloroacetic acid (TCA) on ice. Add TCA to a final concentration of 12% to the CF (3 ml of 100% TCA to the 25 ml sample). Invert the tube several times to mix and put the falcon tube at 4 °C overnight. Ensure the falcon tubes used for precipitation are compatible with organic solvents and high speed centrifugation.
    3. Spin the CF at 15,000 x g for 15 min at 4 °C.
    4. Discard the supernatant carefully to avoid loosing the pellet.
    5. Wash the remaining TCA by adding 5 ml acetone (pre-chilled at 4 °C) slowly along the sides of the tube.
    6. Spin at 15,000 x g for 15 min at 4 °C.
    7. Discard the supernatant very carefully at this stage since the pellet is not adhered tightly to the tube.
    8. Leave the tubes open in a Class II BSC for 10-20 min for faster drying of the pellet. Do not over dry to avoid difficulty in solubilizing of the pellet.  
    9. Resuspend the pellet in 150 µl 1x SDS-PAGE sample buffer. You may need to place tubes in a 65 °C water bath briefly to completely resuspend.
    10. If samples turns yellow due to incomplete removal of TCA, add a few microliters of 1 M Tris-HCl (pH 8) until they turn blue again.
    11. Store sample at -70 °C. Thaw before use. 10 µl of the sample may be enough for SDS-PAGE and western analysis, although the amount will depend upon the amount of the target protein and the sensitivity of the detection method.

Notes

  1. For applications incompatible with TCA precipitations, Centricon Plus-70 centrifugal filter units can be used to concentrate the CF from step B9 as per the manufacturer’s instructions. This can be followed by buffer exchange to desired buffers. Care must be taken to use Centricons with membranes of molecular weight cut-off less than the molecular weight of the target proteins to be analyzed in the CF.

Recipes

  1. 7H9 complete media (1 L)
    7H9 powder 4.7 g
    50% glycerol 4.0 ml
    20% Tween 80 2.5 ml
    Water to 900 ml
    Dissolve 7H9 powder in water and add glycerol and Tween 80
    Adjust the amount of water to give a final volume of 900 ml
    Add 100 ml of OADC for Mtb or 100 ml of ADC for Msmeg and BCG
    Sterilize through 0.22 µM filter and stored at 4 °C (the media can be stored for up to six months. If supplemented with additional additives, like antibiotics or salts, the media should be stored as appropriate for the compounds if they are sensitive to light or degradation)
    Note: Alternatively, autoclave after dissolving 7H9 and glycerol in 897.5 ml water and then supplement with sterile tween-80 and 100 ml of ADC/OADC and store at 4 °C as noted above.
  2. Sauton’s media (1 L)
    Potassium phosphate, monobasic 0.5 g
    L-asparagine monohydrate 4.0 g
    Citric acid monohydrate 2.0 g
    Ferric ammonium citrate 0.05 g 1% Zinc sulfate monohydrate 0.1 ml
    Magnesium sulfate heptahydrate 0.5 g 100% Glycerol 60 ml
    Water to 1 L
    20% Tween 80 2.5 ml
    Dissolve the above components except for the Tween 80 in 900 ml water
    Adjust pH to 7 with 5 N NaOH
    Make up the volume to 1 L with water
    Autoclave, cool, and add 2.5 ml of sterile 20% Tween 80.
    Stored at 4 °C as mentioned above for 7H9 complete media
  3. Chelated Sauton’s media (1 L)
    Potassium phosphate, monobasic 0.5 g
    L-asparagine monohydrate 4.0 g
    Citric acid monohydrate 2.2 g
    Glycerol 60 ml
    20% Tween 80 2.5 ml
    Water to 1 L
    Chelex 100 resin 10 g
    Dissolve the components in water
    Adjust the pH to 7.4 with 5 N NaOH and make up the volume to 1 L with water
    Add 10 g Chelex 100 resin and stir the media for 1-2 days at room temperature
    Sterilize through a 0.22 µM filter unit
    Dissolve 1 g of MgSO4.7H2O in 5 ml water and sterilize it by filtration and add to the sterilized media
    Stored the media at 4 °C as mentioned above for 7H9 complete media
    Defined concentrations of metals like iron, copper and zinc can be added back after chelation.
  4. Protein extraction buffer (10 ml)
    1 M Tris-Cl (pH 7.5) 0.5 ml
    0.5 M EDTA 0.1 ml
    20% SDS 0.3 ml
    Water 9.1 ml
    This can be prepared and stored at 4 °C.
    Immediately prior to use, add Halt Protease Inhibitor cocktail to a final concentration of 1x and keep on ice.
  5. 5x SDS-PAGE sample buffer (50 ml)
    0.5 M Tris (pH 6.8) 12.5 ml
    SDS 5 g
    100% glycerol 25 ml
    Bromophenol blue 0.030 g
    β- mercaptoethanol 500 µl
    Water to 50 ml
    Dissolve SDS in Tris buffer and some water, followed by addition of glycerol and bromophenol blue.
    Mix and make up the volume to 50 ml with water
    Add 500 µl of β- mercaptoethanol
    Freeze in aliquots at -20 °C

Acknowledgments

This protocol was originally used in the published work Mehra et al. (2013). This published work was supported by grants and fellowships from the NIH (R01 AI087682), the Doris Duke Charitable Foundation, the Infectious Disease Society of America, the Michael Saperstein Medical Scholars Research Fund (New York University School of Medicine), Potts Memorial Foundation and the American Society of Microbiology.

References

  1. Mehra, A., Zahra, A., Thompson, V., Sirisaengtaksin, N., Wells, A., Porto, M., Koster, S., Penberthy, K., Kubota, Y., Dricot, A., Rogan, D., Vidal, M., Hill, D. E., Bean, A. J. and Philips, J. A. (2013). Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking. PLoS Pathog 9(10): e1003734.

简介

结核分枝杆菌(Mtb)是结核病的致病因子。由Mtb分泌的蛋白质的分析已经对结核病研究领域感兴趣,因为某些分泌的蛋白质与宿主相互作用以促进毒力,而其他可能是重要的抗原或用作感染的生物标志物。在这里,我们描述了从Mtb或疫苗菌株牛分枝杆菌 - 卡介苗(BCG)制备全细胞提取物(WCE)和短期培养物滤液(CF)的方案(Mehra等人, et al。,2013)。这些都是缓慢生长的分枝杆菌,但是相同的基本程序可以容易地适于分析来自快速生长的分枝杆菌的分泌蛋白,例如耻垢分枝杆菌(Msmeg),其是实验室中常用的非致病物种。可以通过蛋白质印迹分析获得的级分,以检查感兴趣的蛋白质,或者如果抗体不可获得或通过质谱法检查整个分泌蛋白质组。所关注基因的遗传敲除突变体用作阴性对照。另外,应当在CF部分中评估胞质蛋白如分子伴侣GroEL或丙酮酸脱氢酶E2组分sucB(Rv2215/dlaT)的水平,以排除CF中的阳性信号是由于细菌裂解导致的可能性(参见图1)。通过改变菌株的生长条件,这种体外分泌测定法可用于检查改变分泌物组织的条件。我们感谢Magnus Stiegedal提供有关TCA(三氯乙酸)沉淀的有用提示。

关键字:结核分枝杆菌培养, 培养滤液, 全细胞提取物, TCA沉淀, 珠跳动

Feng,J.,Liu,T.,Qin,B.,Zhang,Y.and Liu,X.S。(2012)。 使用MACS识别ChIP-seq富集。 Nat Protoc 7(9):1728-1740
  • Giardine,B.,Riemer,C.,Hardison,RC,Burhans,R.,Elnitski,L.,Shah,P.,Zhang,Y.,Blankenberg,D.,Albert,I.,Taylor, ,W.,Kent,WJ和Nekrutenko,A。(2005)。 Galaxy:用于交互式大规模基因组分析的平台 Genome Res 15(10):1451-1455
  • Lee,K.L.,Buckley,H.R。和Campbell,C.C。(1975)。 一种氨基酸液体合成培养基,用于发展白色念珠菌的菌丝体和酵母形式/em>。 Sabouraudia 13(2):148-153。
  • Znaidi,S.,Nesseir,A.,Chauvel,M.,Rossignol,T.and d'Enfert,C.(2013)。 参与白色念珠菌的两种热休克因子型转录调节因子的综合功能描述形态发生和毒力。 PLoS Pathog 9(8):e1003519。
  • ...
  • Glycerol (Sigma-Aldrich, catalog number: G5516)
  • Albumin-dextrose-catalase (ADC) (BD, catalog number: 212352)
  • Oleic-albumin-dextrose-catalase (OADC) (BD, catalog number: 212351)
  • Potassium phosphate (monobasic) (KH2PO4) (Sigma-Aldrich, catalog number: P9791)
  • L-asparagine monohydrate (Sigma-Aldrich, catalog number: A8381)
  • Citric acid monohydrate (Sigma-Aldrich catalog number: C1909)
  • Ferric ammonium citrate (Sigma-Aldrich catalog number: F5879)
  • Zinc sulfate monohydrate (ZnSO4.H2O) (Sigma-Aldrich, catalog number: 96495)
  • Magnesium sulfate heptahydrate (MgSO4.7H2O) (Sigma-Aldrich, catalog number: 230391)
  • Chelex 100 resin (Bio-Rad Laboratories, catalog number: 142-2822)
  • 100x Halt Protease Inhibitor single use cocktail (Pierce, catalog number: 1860932)
  • 100% trichloroacetic acid (TCA) (pre-chilled prior to use) (Sigma-Aldrich, catalog number: 49010)
  • Acetone (pre-chilled prior to use) (Sigma-Aldrich, catalog number: 32201)
  • Phosphate buffered saline (PBS) (Life Technologies, Gibco®, catalog number: 10010-023)
  • Bromophenol Blue, sodium salt (US Biological, catalog number: 12370)
  • Tris (MP Biomedicals, catalog number: 02194855)
  • Sodium Dodecyl Sulfate (SDS) (US Biological, catalog number: 18220)
  • Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758)
  • β-mercaptoethanol (2-ME) (Sigma-Aldrich, catalog number: M6250)
  • 7H9完整媒体(见配方)
  • Sauton的媒体(见配方)
  • 螯合的sauton的媒体(分枝杆菌生长的另一种基本培养基)(参见食谱)
  • 蛋白质提取缓冲液(参见配方)
  • 5x SDS-PAGE样品缓冲液(见配方)
  • 设备

    1. 高压灭菌器
    2. Steriflip-GV过滤单元(0.22μm孔径)(Millipore,目录号:SE1M179M6)
    3. 20ml注射器(BD,目录号:302830)
    4. 0.22μM注射器过滤单元(33mm)(Millipore,目录号:SLGV033RS)
    5. 一次性无菌过滤系统(1L,0.22μm孔径)(Corning,目录号:09761104)
    6. 0.1mm氧化锆/二氧化硅珠(Bio Spec Products,目录号:11079101z)
    7. 30ml方形培养基瓶(Nalgene ,目录号:NE/2019-0030)
    8. 125毫升方形媒体 瓶(Nalgene ,目录号:NE/2019-0125)
    9. 50ml Falcon管(Corning,目录号430290)
    10. 15ml具有塞密封盖的falcon管(Corning,目录号:430052) 注意:这些50ml管与有机溶剂和高速离心相容。 具有这些功能的猎鹰管可以从不同的供应商使用。
    11. 微管(2ml带O形环的螺帽)(SARSTEDT AG,目录号:72.693)
    12. 分光光度计
    13. 带有用于旋转细菌培养物的摆动转子的离心机(例如,Beckman Coulter,型号:Allegra X-15R;具有SX4750转子的台式离心机)
      注意:
      1. Msmeg和BCG应根据生物安全的实践标准进行处理。
      2. 根据机构实践标准,Mtb文化应在生物安全3级设施中处理。
      3. 离心BCG和Mtb需要适当的气溶胶控制。
    14. 在分枝杆菌培养物离心期间(例如Beckman Coulter,目录号:BK359232),贝克曼气溶胶罐包含气溶胶。
    15. 37℃振荡培养箱
    16. 用于在振荡培养箱中摇动BCG和Mtb液体培养基的气雾剂容纳单元 注意:孵化器应放置在BSB3设施中用于Mtb培养。
    17. 用于CF的CFA沉淀的50ml聚丙烯镰刀的高速离心机(例如具有JLA16.2转子的Beckman Coulter离心机)
    18. 50 ml falcon适配器转子JLA16.2
    19. Bead beater(Bio Spec Products,型号:Minibead beater 16; http://www.biospec.com/product/34/mini_beadbeater/
    20. 带制冷的标准台式离心机
    21. 用于Eppendorf管的加热块(设置为95℃
    22. pH计

    首字母缩略词

    1. Mtb:结核分枝杆菌
    2. BCG:牛分枝杆菌 Calmatte-Guérin
    3. Msmeg: Mycobacterium smegmatis
    4. WCE:全细胞提取物
    5. CF:培养滤液
    6. DlaT:分枝杆菌的Rv2215 /丙酮酸脱氢酶E2组分sucB蛋白
    7. TCA:三氯乙酸
    8. BSC:生物安全柜
    9. OD <600>:波长为600nm的吸光度或光密度

    程序

    1. 分枝杆菌培养物的接种和生长
      1.  接种来自冷冻股票的分枝杆菌的液体培养物,制备 从对数期中期分枝杆菌培养物(OD 600在0.5至1.0之间) 其在-80℃下在18%甘油中冷冻。
        1. 对于接种, 需要牢记分枝杆菌物种的倍增速率。   Msmeg大约每3小时翻倍,而BCG和Mtb翻倍 大约在37℃下在7H9完全培养基中每20小时。
        2. 的 将培养物接种在30ml正方形的10ml 7H9完全培养基中 媒体瓶。 如果需要,包括适当的抗生素。例如,抗生素可以适当的浓度包括在选择中   质粒时,生长转化的分枝杆菌菌株。
        3. 的 将分枝杆菌培养物在37℃下缓慢振荡在37℃下孵育 速度为90rpm。 BCG和Mtb培养物置于气溶胶容纳中 在摇动培养箱中
        4. 对于Msmeg,接种200微升从 甘油储备液并生长过夜至饱和。 稀释过夜 培养1:40成新鲜7H9完全培养基,并生长至对数中期。 对于BCG和Mtb,从冷冻储存物接种0.5ml。 它会采取 几天(〜4天)使培养物达到对数中期 精确时间取决于初始接种物密度和生长 条件。
      2. 测量培养物的OD 600。 确定 需要达到至少0.25的最终起始OD 600的培养体积 当重悬于25ml Sauton's培养基[培养体积(ml)= 25 ml×所需实验培养物的OD 600(例如0.25)/OD 600)。
      3. 将这一体积的培养基转移到15毫升falcon管和 通过在室温下以1500×g离心7分钟来沉淀细菌细胞   温度。 取出上清液,并在PBS中重悬沉淀。 再次在1,500xg离心7分钟。 这个洗涤步骤很重要 去除从7H9完全培养基携带的白蛋白。  
        注意:
        1. 根据我们对离心Mtb的实践的制度标准 和BCG培养物,将falcon管置于离心适配器中 放在Aerosolve 罐中(在Equipment,第14项中提到)   II类生物安全柜(BSC II)。
        2. 这些从罩中移出并装载到转子中并离心。
        3. 旋转后,将具有猎鹰管的整个Aerosolve 罐带回BSC II罩并打开。
        4. 此程序包含离心过程中产生的气溶胶。 Mtb文化应在BSL3设施中处理。
      4. 除去上清液,并重悬在1毫升的细菌沉淀 Sauton的媒体。 转移到125毫升方瓶介质瓶 24ml Sauton's培养基(在Sauton's中包括合适的抗生素 媒体)。 在振荡培养箱中37℃孵育。
        注意:
        1. Sauton的培养基是其中分枝杆菌生长比它们在7H9完全培养基中更慢的最小培养基。
        2. 或者,如果金属离子对分泌的影响是有效的 研究,可以使用螯合的Sauton's介质作为基本介质和 添加定义的金属离子浓度。
      5. 监视OD 600 文化。 将培养物培养至0.5至0.8OD 600之间。 大约需要6小时   对于Msmeg和48小时的BCG和Mtb这个程序可以修改为 以较低的密度接种细菌并允许它们生长 更长的时间,这取决于实验的目的。

    2. 收获细菌培养物和WCE的制备
      1. 如上所述在Sauton's培养基中孵育后,测定OD 600。
      2. 计算对应于5OD 600单位的体积。 例如,if 培养物的OD 600为0.5,则10 ml将对应于5 OD 单位(所需体积= 5/OD 600实验培养物)
      3. 转移5 OD 600培养物到15ml falcon管(用于   准备WCE)。 将剩余的培养物转移到50ml的猎鹰 管(用于从CF沉淀蛋白质)。 离心两者 管在2,000×g下在4℃下5分钟。
      4. 合并上清液 从15和50毫升猎鹰进入一个未使用的50毫升falcon管和   存储在冰上,直到你准备好进一步处理它(见下文, 在步骤C)中制备CF。  
      5. 通过在2,000×g,5分钟,4℃下重悬和离心,用5ml PBS洗涤沉淀在15ml Falcon中。
      6. 弃去上清液,并再次悬浮在1ml PBS中的沉淀 并将其转移到具有O形环的2ml Sarstedt管中 (用于在珠粒搅拌期间容纳气溶胶)。
      7. 旋转上 台式离心机在3000×g下离心5分钟,弃去上清液。 至 沉淀加入300μl含蛋白酶的蛋白提取缓冲液 抑制剂。 通过pippettount重悬沉淀。
      8. 分配100 μl的0.1mm氧化锆/二氧化硅珠倒入1.7ml eppendorf中并加入 到含有颗粒的Sarstedt管
      9. 使用珠 搅拌器包含在II级生物安全柜内(请阅读 珠子搅拌器手册的操作程序和也看到链接 在设备中提供,项目19),珠粒在3.450下敲打样品1分钟   振荡/min并将其置于冰上2分钟以冷却。 重复此操作 两次。 这个样本是WCE。 将裂解液全速离心2分钟   min在微量离心机中以在打开管之前沉淀泡沫。
        注意:
        1. 替代的珠子搅拌器可以具有不同的速度,因此用户 应优化珠打(速度和时间)以最大程度裂解 分枝杆菌颗粒。
        2. 可以通过在用于菌落形成单位的珠打之前和之后对细菌进行电镀来建立裂解的程度。
      10. 对于SDS-PAGE,向Sarstedt添加60μl的5×SDS-PAGE样品缓冲液 然后通过倒置轻轻混合。 在95℃孵育5分钟。 的 加热步骤有助于样品变性和灭菌 分枝杆菌裂解物。 分枝杆菌WCE可以 储存于-80℃。
        注意:Mtb的WCE通常可以传输 出来的BSL3设施在这一步只要是一致的   机构的实践标准。
      11. 使用前,在室内解冻   温度,然后在95℃加热5分钟。 经常有20μl的WCE   足以进行SDS-PAGE和Western分析,尽管这个量会   取决于目标蛋白水平和敏感性 检测方法。

    3. TCA沉淀的CF
      1. 使用25ml注射器将来自步骤B9的培养上清通过0.22μM过滤器过滤到新鲜的50ml falcon管中。
        注意:
        1. 或者,可以使用50ml Steriflip-GV通过真空过滤 过滤单位(0.22μM)以从上清液中除去任何细菌。
        2. 这可以根据上清液的浊度进行两次。
        3. 根据标准,也可能需要双重过滤 操作程序从BSL3传送CF 设施。
      2. 在冰上预冷三氯乙酸(TCA)。 加 TCA至终浓度为12%至CF(3ml的100%TCA中 25ml样品)。 倒置管几次混合和放猎鹰 在4℃过夜。 确保猎鹰管用于沉淀 与有机溶剂和高速离心相容
      3. 在4℃下将CF在15,000×g下旋转15分钟。
      4. 小心弃去上清液,以免丢失沉淀。
      5. 通过沿管的侧面缓慢加入5 ml丙酮(在4°C预冷),清洗剩余的TCA。
      6. 在4℃下以15,000ing/g旋转15分钟。
      7. 在这个阶段非常小心地弃去上清液,因为颗粒不紧密地粘附到管上。
      8. 将管在II级BSC中打开10-20分钟以更快干燥   的颗粒。 不要过度干燥,以免溶解困难 颗粒。  
      9. Resuspend the pellet in 150 µl 1x SDS-PAGE sample buffer. You may need to place tubes in a 65 °C water bath briefly to completely resuspend.
      10. If samples turns yellow due to incomplete removal of TCA, add a few microliters of 1 M Tris-HCl (pH 8) until they turn blue again.
      11. Store sample at -70 °C. Thaw before use. 10 µl of the sample may be enough for SDS-PAGE and western analysis, although the amount will depend upon the amount of the target protein and the sensitivity of the detection method.

    笔记

    1. 对于与TCA沉淀不相容的应用,可以使用Centricon Plus-70离心过滤器单元按照制造商的说明书浓缩步骤B9的CF。 这之后可以进行缓冲液交换至所需的缓冲液。 必须小心使用具有小于在CF中待分析的目标蛋白质的分子量的截留分子量的膜的Centricons。

    食谱

    1. 7H9完全培养基(1L)
      7H9粉末4.7g
      50%甘油4.0ml
      20%Tween 80 2.5ml
      水至900 ml
      将7H9粉末溶解在水中,并加入甘油和吐温80 调整水量,使最终体积为900 ml
      加入100ml OADC用于Mtb或100ml ADC用于Msmeg和BCG
      通过0.22μM过滤器灭菌,并在4°C下储存(培养基可以保存长达6个月。如果补充了其他添加剂,如抗生素或盐,如果对光敏感,培养基应该适合于化合物储存或退化)
      注意:或者,将7H9和甘油溶解在897.5ml水中,然后用无菌tween-80和100ml ADC/OADC补充,并如上所述在4℃下储存,高压灭菌。
    2. Sauton的媒体(1升)
      磷酸钾,一碱价0.5g
      L-天冬酰胺一水合物4.0g
      柠檬酸一水合物2.0g
      柠檬酸铁铵0.05g 1%硫酸锌一水合物0.1ml
      硫酸镁七水合物0.5g 100%甘油60ml
      水至1 L
      20%Tween 80 2.5ml
      将上述组分(吐温80除外)溶于900ml水中
      用5N NaOH将pH调节至7 用水将体积补至1升,
      高压灭菌,冷却,并加入2.5 ml无菌20%吐温80.
      如上所述在4℃下储存7H9完全培养基
    3. Chelated Sauton的媒体(1升)
      磷酸钾,一碱价0.5g
      L-天冬酰胺一水合物4.0g
      柠檬酸一水合物2.2g
      甘油60 ml
      20%Tween 80 2.5ml
      水至1升
      Chelex 100树脂10g 将组分溶解在水中
      用5N NaOH调节pH至7.4,用水补足体积至1L 加入10g Chelex 100树脂,并在室温下搅拌培养基1-2天 通过0.22μM过滤器单元灭菌
      将1g MgSO 4·7H 2 O 7H 2 O溶解在5ml水中,通过过滤对其进行灭菌,并加入到灭菌培养基中。
      将培养基保存在4℃下,如上所述用于7H9完全培养基
      可以在螯合后加入限定浓度的金属,如铁,铜和锌
    4. 蛋白提取缓冲液(10ml)
      1M Tris-Cl(pH7.5)0.5ml
      0.5M EDTA 0.1ml
      20%SDS 0.3ml
      水9.1ml
      这可以在4℃下制备和储存。
      在使用前,立即加入Halt蛋白酶抑制剂混合物至最终浓度为1x,并保持冰上
    5. 5×SDS-PAGE样品缓冲液(50ml) 0.5M Tris(pH6.8)12.5ml
      SDS 5 g
      100%甘油25ml
      溴酚蓝0.030g
      β-巯基乙醇500μl
      水至50ml
      将SDS溶于Tris缓冲液和一些水中,然后加入甘油和溴酚蓝 混合并用水补足体积至50ml 加入500μlβ-巯基乙醇 以等分试样在-20°C下冻融

    致谢

    该协议最初用于公开的工作Mehra等人(2013)。 这项出版的工作得到了NIH(R01 AI087682),Doris Duke慈善基金会,美国传染病学会,Michael Saperstein医学学者研究基金(纽约大学医学院),Potts纪念基金会和 美国微生物学会。

    参考文献

    1. Mehra,A.,Zahra,A.,Thompson,V.,Sirisaengtaksin,N.,Wells,A.,Porto,M.,Koster,S.,Penberthy,K.,Kubota,Y.,Dricot, Rogan,D.,Vidal,M.,Hill,DE,Bean,AJ和Philips,JA(2013)。 结核分枝杆菌型VII分泌的效应物EsxH靶向宿主ESCRT以损害运输。 a> PLoS Pathog 9(10):e1003734。
    • 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: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
    1. Mehra, A. and Philips, J. A. (2014). Analysis of Mycobacterial Protein Secretion . Bio-protocol 4(12): e1159. DOI: 10.21769/BioProtoc.1159.
    2. Mehra, A., Zahra, A., Thompson, V., Sirisaengtaksin, N., Wells, A., Porto, M., Koster, S., Penberthy, K., Kubota, Y., Dricot, A., Rogan, D., Vidal, M., Hill, D. E., Bean, A. J. and Philips, J. A. (2013). Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking. PLoS Pathog 9(10): e1003734.
    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。