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Extraction and Purification of Mycobacterial Mycolic Acids
分枝杆菌霉菌酸的提取和纯化   

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Abstract

Mycolic acids are major long-chain fatty acids, containing up to 80-90 carbon atoms that represent essential components of the mycobacterial cell wall (Pawelczyk and Kremer, 2014). Each mycobacterial species possesses a specific mycolic acid profile characterized by various chemical modifications that decorate the lipid. Mycolic acids play a critical role in the architecture and impermeability of the cell envelope, hence the natural resistance of mycobacteria to most antibiotic treatments. They are also key determinants of virulence in pathogenic species, including Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis. In addition, they are known as the primary target of several first-line and second-line antitubercular drugs. Thus, the unique enzymes involved in the mycolic acid biosynthetic pathway represent an attractive reservoir of targets for future chemotherapy whose developments are particularly warranted in the context of multi-drug-resistant and extensively-drug-resistant strains of M. tuberculosis. Herein, we describe a protocol to extract the mycolic acids from mycobacteria. Purification of the various subspecies may be particularly useful for subsequent structural studies involving mass spectrometry or NMR. The qualitative and quantitative biochemical characterization of the mycolic acid pattern by thin layer chromatography can be used to address how drugs alter mycolic acid biosynthesis (Alahari et al., 2007, Hartkoorn et al., 2012), to study the phenotypes of genetically modified mutants affected in this metabolic pathway (Bhatt et al., 2007) or to unravel new mycolic acid regulatory mechanisms (Vilcheze et al., 2014). The same protocol can be applied to all mycobacteria, including environmental and pathogenic species.

Keywords: Mycobacterium(结核分枝杆菌), Mycolic acid(分枝菌酸), Cell wall(细胞壁), Thin layer chromatography(薄层色谱法), Lipid(脂质)

Materials and Reagents

  1. Mycobacterial cultures including Mycobacterium tuberculosis, BCG, M. smegmatis and M. abscessus but can be extended to any other species.
  2. Tyloxapol (C17H30O4) (Sigma-Aldrich, catalog number: T8761 )
  3. Glycerol (C3H8O3, 99.5%) (Euromedex, catalog number: 50405 )
  4. Potassium diphosphate (K2HPO4) (Merck KGaA, catalog number: 1.05104 )
  5. L-asparagine monohydrate (C4H10N2O4, ≥99%) (Sigma-Aldrich, catalog number: A8381 )
  6. Citric acid (C6H8O7, 99%) (Sigma-Aldrich, catalog number: C0759 )
  7. Ferric ammonium citrate (C7H13FeNO7) (Sigma-Aldrich, catalog number: F5879 )
  8. Zinc sulfate heptahydrate (ZnSO4.7H2O, ≥99%) (Sigma-Aldrich, catalog number: Z4750 )
  9. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M7506 )
  10. Tetrabutyl ammonium hydroxyde (TBAH) (C16H37NO, 40% solution) (Sigma-Aldrich, catalog number: 178780 )
  11. Dichloromethane (CH2Cl2, ≥99.8%) (Carlo Erba, catalog number: 412622000 )
  12. Iodomethane (CH3I, 99%) (Sigma-Aldrich, catalog number: I850-7 )
  13. Diethylether (CH3CH2OCH2CH3, 99.5%) (Carlo Erba, catalog number: 447521 )
  14. Hexane [CH3(CH2)4CH3, pure isomeric mixture] (Carlo Erba, catalog number: 339852 )
  15. Ethyl acetate (CH3COOC2H5, 99.9%) (Carlo Erba, catalog number: 448251 )
  16. Molybdophosphoric acid hydrate (H5Mo12O41P)  (Sigma-Aldrich, catalog number P7390 )
  17. Rhodamine 6G (C28H31ClN2O3, Dye content ~95 %) (Sigma-Aldrich, catalog number R4127 )
  18. Middlebrook 7H9 broth (Difco, catalog number: 271310 )
  19. Sauton’s media (see Recipes)

Equipment

  1. Aluminium Silica gel TLC plates 60F254 (Merck KGaA, catalog number: 1.05554.0001 )
  2. Plastic Silica gel TLC plates 60F254 (Merck KGaA, catalog number: 1.05735.0001 )
  3. Round-bottom borosilicate glass tubes (Corning, catalog number: 99449-16 ) with phenolic screw caps (Corning, catalog number: 9998-15 )
  4. 10 µl capillary tubes Ringcaps (Dutscher Scientific, catalog number: 0 90512 )
  5. Spectrophotometer
  6. 75 cm2 tissue culture flasks (Sarstedt, catalog number: 83.1813 )
  7. 37 °C incubator or 30 °C incubator for growing mycobacteria such as M. marinum
    Notes: Atypical mycobacteria and BCG should be handled according to institutional standards of practice for biosafety whereas M. tuberculosis cultures should be handled in biosafety level 3 facility.
  8. 50 ml conical Corning CentriStart tubes (Corning, catalog number: 430828 )
  9. 15 ml conical Corning CentriStart tubes (Corning, catalog number: 430790 )
  10. Centrifuge with swinging bucket rotor for spinning down bacterial cultures (for example, Prolabo, model: HR340 with the 15 ml and 50 ml adaptors)
  11. pH meter
  12. Ultrasonic waterbath (Bandelin Sonorex, model: RK102H )
  13. Stuart Rotator SB3
  14. Stuart block heater (Stuart, model: SBH200D )
  15. TLC developing tank
  16. Zymark TurboVap evaporator (Biotage, catalog number: C133718 )
  17. Handheld UV lamp VL-6-LC 365 nm/254 nm
  18. 1800W Heat gun (Power Performance, model: PHG1800T2 )
  19. Reagent Sprayer, all glass, with 100 ml Erlenmeyer flask (CAMAG, catalog number: 022.6100 )
  20. TLC Spray Cabinet (CAMAG, catalog number: 0 22.623 0)

Procedure

  1. Inoculation and growth of mycobacterial cultures
    1. Inoculate liquid cultures (in either Sauton’s broth or Middlebrook 7H9 broth) of mycobacteria from frozen stocks, prepared from mid-log phase mycobacterial cultures (OD600 = 0.8 to 1.0) that were frozen in 50% glycerol at -80 °C. For inoculation, the doubling rate of the mycobacterial species needs to be kept in mind. M. smegmatis doubles approximately every 3-4 h, whereas BCG and M. tuberculosis double approximately every 24 h at 37 °C.
    2. These pre-cultures are inoculated in 25 ml Sauton’s broth medium (or Middlebrook 7H9 broth) in 75 cm2 tissue culture flasks. Include appropriate antibiotics if required for maintaining a plasmid when growing a transformed mycobacterial strain.
    3. The mycobacterial cultures are incubated at 37 °C with no or slow shaking. Measure the OD600 of the culture and harvest exponentially growing mycobacteria (OD600 = 0.8 to 1.0) by centrifugation at 3,000 rpm for 10 min at room temperature. Discard the supernatant.
    4. Resuspend the pellet in 5 ml of water and transfer the bacterial suspension in 15 ml conical plastic tubes. Pellet the bacterial cells again by centrifugation. This wash step is important to remove the remaining traces of medium. The bacterial pellet is then transferred to a round-bottom glass tube prior to lipid hydrolysis. All subsequent procedures will be performed in glass tubes due to the presence of organic solvents.

  2. Fatty acid/mycolic acid extraction
    1. Resuspend the bacterial pellet in 2 ml of tetrabutyl ammonium hydroxyde (TBAH) and incubate overnight at 100 °C using a heating block. This step allows complete fatty/mycolic acid hydrolysis.
    2. The next day, let the tube cool down at room temperature and proceed with methyl esterification of the fatty/mycolic acids: To the 2 ml of TBAH mixture, add 4 ml CH2Cl2, 300 μl CH3I and 2 ml water. Mix the tube for 1 h at room temperature on a rotator.
    3. Centrifuge at 3,500 rpm for 10 min at room temperature. This will allow to separating the mixture in two phases: A lower organic phase containing the lipids and an upper aqueous phase. Discard the upper phase to waste.
    4. Add 4 ml of water, mix the tube for 15 min and spin down at 3,500 rpm for 10 min at room temperature. Repeat this wash step 2 more times and remove the upper phase to waste.
    5. Dry the lower organic phase in the same tube under a stream of nitrogen using a TurboVap evaporator. Evaporation of the organic phase takes around 30 min.
    6. To the dried residue, add 3 ml diethylether and sonicate in a waterbath for 10 min at room temperature.
    7. Centrifuge at 3,500 rpm for 10 min at room temperature and, using a Pasteur pipette, transfer the fatty acid/mycolic acid methyl esters in a new glass tube.
    8. Evaporate the diethylether under a stream of nitrogen and resuspend the residue in 100-200 µl CH2Cl2.

  3. Thin layer chromatography
    1. Load 10-30 µl on an aluminium TLC plate using 10 µl capillary tubes (Figure 1).


      Figure 1. Loading the lipid samples with a capillary tube directly at the origin of the TLC plate

    2. Fatty acid methyl esters (FAMES) and mycolic acid methyl esters (MAMES) are separated by thin layer chromatography in a TLC tank using hexane/ethyl acetate (19:1, v/v). When the solvent front reaches the top of the TLC plate, the plate is taken out, dried very briefly and placed again in the tank for a second round of migration. This allows to better separate the various mycolic acid subspecies.
    3. Spray the TLC plate with 5% ethanolic molybdophosphoric acid using a reagent sprayer in a TLC spray cabinet (Figure 2).


      Figure 2. Homogenous spraying of the TLC plate with molybdophosphoric acid in a spray cabinet

    4. Lipids are revealed following charring using a heat gun, which emits a stream of very hot air. Bands corresponding to the different mycolic acids will appear as represented in Figure 3.

  4. Purification of the mycolic acid subspecies
    1. Mycolic acid methyl esters are loaded on preparative plastic TLC plates and separated, as described above.
    2. Spray the plate with 0.01% ethanolic Rhodamine 6G and visualize the mycolic acid subspecies using a handheld UV lamp.
    3. Scrape off the bands corresponding to the individual mycolic acid subspecies and put the lipid-loaded silica powder into a glass tube.
    4. Extract from mycolic acid with diethyl ether as mentioned above (steps B6-8).
    5. If necessary, resolve again the lipids individually on new preparative TLC plates and repeat the whole procedure.
    6. Evaporate the diethylether under a stream of nitrogen and resuspend the lipids in 100-200 µl CH2Cl2.
    7. Assess purity of the mycolic acid methyl esters on a standard TLC plate as described in steps C1-4.

Representative data


Figure 3. Mycolic acid profile of various mycobacterial species. Cultures of M. tuberculosis CDC1551, BCG Pasteur, M. smegmatis mc2155 and M. abscessus CIP104536T were grown in Sauton’s medium. FAMEs and MAMEs were extracted and analyzed by one-dimensional TLC using hexane/ethyl acetate (19:1, v/v; 2 runs) and revealed by spraying the plate with molybdophosphoric acid followed by charring. α-, m, k, corresponding respectively to alpha-, methoxy- and keto-mycolic acids are characteristic of M. tuberculosis. BCG Pasteur lacks methoxy mycolic acids. M. abscessus possesses only α- and α‘-mycolic acids shared with M. smegmatis which produces also epoxy-myolic acids (e). FAME, fatty acid methyl ester; MAME, mycolic acid methyl ester.

Notes

For all steps involving organic solvents, use glass pipettes and tubes and work under a chemical hood.

Recipes

  1. Sauton’s media (1 L)
    Dipotassium phosphate
    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
    5% tyloxapol
    5 ml
    Water to 1 L
    Dissolve the above components
    Adjust pH to 7-7.2 with 5 N NaOH
    Make up the volume to 1 L with water
    Autoclave for 10 min
    Stored at room temperature

Acknowledgments

The authors wish to thank Vaincre la Mucoviscidose for funding CMD. This protocol was adapted from a previous work by This protocol was adapted from a previous work by Besra (1998).

References

  1. Alahari, A., Trivelli, X., Guérardel, Y., Dover, L. G., Besra, G. S., Sacchettini, J. C., Reynolds, R. C., Coxon, G. D. and Kremer, L. (2007). Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria. PLoS One 2(12): e1343.
  2. Besra, G. S. (1998). Preparation of cell-wall fractions from mycobacteria. Methods Mol Biol 101: 91-107.
  3. Bhatt, A., Fujiwara, N., Bhatt, K., Gurcha, S. S., Kremer, L., Chen, B., Chan, J., Porcelli, S. A., Kobayashi, K., Besra, G. S. and Jacobs, W. R., Jr. (2007). Deletion of kasB in Mycobacterium tuberculosis causes loss of acid-fastness and subclinical latent tuberculosis in immunocompetent mice. Proc Natl Acad Sci U S A 104(12): 5157-5162.
  4. Hartkoorn, R. C., Sala, C., Neres, J., Pojer, F., Magnet, S., Mukherjee, R., Uplekar, S., Boy-Rottger, S., Altmann, K. H. and Cole, S. T. (2012). Towards a new tuberculosis drug: pyridomycin – nature’s isoniazid. EMBO Mol Med 4 (10): 1032-1042.

简介

霉菌酸是主要的长链脂肪酸,含有高达80-90个碳原子,代表分枝杆菌细胞壁的基本组分(Pawelczyk和Kremer,2014)。每种分枝杆菌物种具有特征性霉菌酸分布,其特征在于装饰脂质的各种化学修饰。霉菌酸在细胞包膜的结构和不可渗透性中起关键作用,因此分枝杆菌对大多数抗生素治疗的天然抗性。它们也是在致病物种中的毒力的关键决定因素,包括结核分枝杆菌(结核分枝杆菌),结核病的致病因子。此外,它们被认为是几种一线和二线抗结核药物的主要靶点。因此,参与霉菌酸生物合成途径的独特的酶代表了用于未来化学疗法的靶的有吸引力的储库,其发展在M的多药耐药和广泛耐药菌株的背景下是特别保证的。肺结核。在这里,我们描述了从分枝杆菌中提取霉菌酸的方案。各种亚种的纯化对于涉及质谱或NMR的后续结构研究可能是特别有用的。通过薄层色谱的霉菌酸模式的定性和定量生物化学表征可用于解决药物如何改变霉菌酸生物合成(Alahari等人,2007,Hartkoorn等人, (Bhatt等人,2007)或解开新的霉菌酸调节机制(Vilcheze等人,2012),以研究在该代谢途径中受影响的遗传修饰的突变体的表型(Bhatt等人, ,2014)。相同的方案可以应用于所有分枝杆菌,包括环境和致病物种。

关键字:结核分枝杆菌, 分枝菌酸, 细胞壁, 薄层色谱法, 脂质

材料和试剂

  1. 结核菌培养物,包括结核分枝杆菌,BCG, smegmatis 和 M。 脓肿,但可以扩展到任何其他物种
  2. (Sigma-Aldrich,目录号:T8761)
    (Sigma-Aldrich,目录号:
  3. 甘油(C 3 H 8 O 3,99.5%)(Euromedex,目录号:50405)
  4. 二磷酸钾(K 2 HPO 4)(Merck KGaA,目录号:1.05104)
  5. L-天冬酰胺一水合物(C 4 H 10 NH 2 N 2 O 4,≥99%)(Sigma-Aldrich ,目录号:A8381)
  6. 柠檬酸(C 6 H 8 O 7,99%)(Sigma-Aldrich,目录号:C0759)
  7. 柠檬酸铁(C 7 H 13,FeNO 7)(Sigma-Aldrich,目录号:F5879)
  8. 硫酸锌七水合物(ZnSO 4·7H 2 O,≥99%)(Sigma-Aldrich,目录号:Z4750)
  9. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M7506)
  10. 四丁基铵氢氧化物(TBAH)(C 16 H 37 NO,40%溶液)(Sigma-Aldrich,目录号:178780)
  11. 二氯甲烷(CH 2 Cl 2,≥99.8%)(Carlo Erba,目录号:412622000)
  12. 碘甲烷(CH 3 I,99%)(Sigma-Aldrich,目录号:I850-7)
  13. 二乙醚(CH 3 CH 2 OCH 2 CH 2 CH 3,99.5%)(Carlo Erba,目录号:447521 )
  14. 己烷[CH 3(CH 2)4] CH 3 CH 3,纯异构体混合物](Carlo Erba,目录号 :339852)
  15. 乙酸乙酯(CH 3 COCO 2 H 5,99.9%)(Carlo Erba,目录号:448251)
  16. 钼磷酸水合物(H 5 Mo 12 P 12 O 41 P) (Sigma-Aldrich,目录号P7390)
  17. 罗丹明6G(C 28 H 31 ClCl 2 Sub 2 O 3,染料含量〜95%)(Sigma- Aldrich,目录号R4127)
  18. Middlebrook 7H9肉汤(Difco,目录号:271310)
  19. Sauton的媒体(见Recipes)

设备

  1. 铝硅胶TLC板60F 254(Merck KGaA,目录号:1.05554.0001)
  2. 塑料硅胶TLC板60F 254(Merck KGaA,目录号:1.05735.0001)
  3. 带有酚醛螺帽的圆底硼硅酸盐玻璃管(Corning,目录号:99449-16)(Corning,目录号:9998-15)
  4. 10μl毛细管环帽(Dutscher Scientific,目录号:090512)
  5. 分光光度计
  6. 75cm 2组织培养瓶(Sarstedt,目录号:83.1813)
  7. 37℃培养箱或30℃培养箱中用于生长分枝杆菌如M。 marinum
    注意:非典型分枝杆菌和BCG应根据生物安全的实践标准进行处理,而 结核病 文化应在生物安全3级机构处理。
  8. 50ml锥形Corning CentriStart管(Corning,目录号:430828)
  9. 15ml锥形Corning CentriStart管(Corning,目录号:430790)
  10. 带有用于旋转细菌培养物的摆动转子的离心机(例如,Prolabo,型号:具有15ml和50ml适配器的HR340)
  11. pH计
  12. 超声波水浴(Bandelin Sonorex,型号:RK102H)
  13. Stuart Rotator SB3
  14. Stuart块加热器(Stuart,型号:SBH200D)
  15. TLC显影槽
  16. Zymark TurboVap蒸发器(Biotage,目录号:C133718)
  17. 手持式紫外灯VL-6-LC 365nm/254nm
  18. 1800W热风枪(Power Performance,型号:PHG1800T2)
  19. 试剂喷雾器,所有玻璃,具有100ml锥形瓶(CAMAG,目录号:022.6100)
  20. TLC Spray Cabinet(CAMAG,目录号:022.6230)

程序

  1. 分枝杆菌培养物的接种和生长
    1. 接种从冷冻于50%甘油中的对数期分枝杆菌培养物(OD 600 = 0.8至1.0)制备的来自冷冻储存物的分枝杆菌的液体培养物(在Sauton肉汤或Middlebrook 7H9肉汤中) -80℃。对于接种,需要记住分枝杆菌物种的倍增速率。 M。耻骨炎大约每3-4小时加倍,而BCG和 M。结核病在37℃下大约每24小时一次。
    2. 将这些预培养物接种在75cm 2组织培养烧瓶中的25ml Sauton's肉汤培养基(或Middlebrook 7H9肉汤)中。如果需要,包括适当的抗生素以在生长转化的分枝杆菌菌株时维持质粒。
    3. 将分枝杆菌培养物在37℃下孵育,没有或缓慢摇动。通过在室温下以3,000rpm离心10分钟,测量培养物的OD 600并收获指数生长的分枝杆菌(OD 600 = 0.8至1.0)。弃去上清液。
    4. 将沉淀重悬于5ml水中,并将细菌悬浮液转移到15ml锥形塑料管中。通过离心再次沉淀细菌细胞。该洗涤步骤对于除去残留的培养基痕量是重要的。然后在脂质水解之前将细菌沉淀转移到圆底玻璃管中。由于有机溶剂的存在,所有后续程序将在玻璃管中进行
  2. 脂肪酸/霉菌酸提取
    1. 将细菌沉淀重悬于2ml四丁基氢氧化铵(TBAH)中,并使用加热块在100℃温育过夜。该步骤允许完全脂肪/霉菌酸水解
    2. 第二天,使管在室温下冷却并进行脂肪酸/霉菌酸的甲基酯化:向2ml TBAH混合物中加入4ml CH 2 Cl 2溶液, /300μlCH 3 I和2ml水。在室温下在旋转器上混合管1小时
    3. 在室温下以3500rpm离心10分钟。这将允许在两个相中分离混合物:含有脂质的下部有机相和上部水相。丢弃上层相,浪费。
    4. 加入4ml水,混合管15分钟,并在室温下以3500rpm离心10分钟。重复该洗涤步骤2次,并将上层相除去。
    5. 使用TurboVap蒸发器在氮气流下在相同管中干燥下部有机相。 有机相的蒸发约需30分钟。
    6. 向干燥的残余物中加入3ml乙醚并在水浴中在室温下超声处理10分钟。
    7. 在室温下以3500rpm离心10分钟,使用巴斯德移液管将脂肪酸/霉菌酸甲酯转移到新的玻璃管中。
    8. 在氮气流下蒸发二乙醚并将残余物重悬在100-200μlCH 2 Cl 2溶液中。

  3. 薄层色谱
    1. 使用10μl毛细管在铝TLC板上加载10-30 μl(图1)。

      图1.用毛细管直接在TLC板的原点装载脂质样品

    2. 在TLC罐中使用己烷/乙酸乙酯(19:1,v/v)通过薄层色谱法分离脂肪酸甲酯(FAMES)和霉菌酸甲酯(MAMES)。当溶剂前沿到达TLC板的顶部时,取出板,非常短暂地干燥,并再次放置在罐中用于第二轮迁移。这允许更好地分离各种霉菌酸亚种
    3. 使用试剂喷雾器在TLC喷雾箱(图2)中用5%的磷钼酸乙醇溶液喷涂TLC板(图2)。


      图2.在喷雾箱中用磷钼酸均匀喷涂TLC板

    4. 使用热风枪(hot gun)炭化后显示脂质,其发射非常热的空气流。 对应于不同霉菌酸的条带将如图3所示。

  4. 霉菌酸亚种的纯化
    1. 霉菌酸甲酯加载到制备性塑料TLC板上并分离,如上所述。
    2. 用0.01%乙醇罗丹明6G喷洒板并使用手持式UV灯显现霉菌酸亚种。
    3. 刮除对应于各个霉菌酸亚种的条带,并将载有脂质的二氧化硅粉末置于玻璃管中。
    4. 如上所述用二乙醚从霉菌酸中提取(步骤B6-8)
    5. 如果需要,在新制备TLC板上单独分离脂质并重复整个程序。
    6. 在氮气流下蒸发二乙醚并将脂质重悬于100-200μlCH 2 Cl 2溶液中。
    7. 在标准TLC板上评估霉菌酸甲酯的纯度,如步骤C1-4中所述

代表数据


图3.各种 分枝杆菌 物种的霉菌酸分布。 M的文化。 结核病 CDC1551,BCG Pasteur,M。 smegmatis mc 2 155和 M。 脓肿</em> CIP104536< sup>在Sauton培养基中生长。提取FAME和MAME,并通过一维TLC使用己烷/乙酸乙酯(19:1,v/v; 2次运行)进行分析,并通过用钼磷酸喷射板显示,然后炭化。 α-,m,k分别对应于α-,甲氧基和酮 - 霉菌酸是M的特征。肺结核。 BCG巴斯德缺乏甲氧基霉菌酸。 M。脓肿仅具有与m共享的α-和α'-霉菌酸。耻垢分泌物,其还产生环氧 - 丝氨酸(e)。 FAME,脂肪酸甲酯; MAME,霉菌酸甲酯。

笔记

对于涉及有机溶剂的所有步骤,使用玻璃移液管和管,并在化学罩下工作。

食谱

  1. Sauton的媒体(1升)
    磷酸二钾
    0.5克
    L-天冬酰胺一水合物 4.0克
    柠檬酸一水合物
    2.0克
    柠檬酸铁铵
    0.05 g
    1%硫酸锌一水合物 0.1 ml
    硫酸镁七水合物
    0.5克
    100%甘油 60 ml
    5%特氟沙平
    5 ml
    水至1 L
    溶解上述成分
    用5N NaOH将pH调节至7-7.2 用水将体积补至1升,
    高压灭菌10分钟
    在室温下储存

致谢

作者希望感谢Vaincre la Mucoviscidose为CMD提供资金。该协议改编自以前的工作,该协议改编自Besra(1998)的以前的工作。

参考文献

  1. Alahari,A.,Trivelli,X.,Guérardel,Y.,Dover,L.G.,Besra,G.S.,Sacchettini,J.C.,Reynolds,R.C.,Coxon,G.D.and Kremer, 硫脲酮,一种抑制细胞壁分枝菌酸的环丙烷化的抗结核药物在分枝杆菌中。 PLoS One 2(12):e1343。
  2. Besra,G.S。(1998)。 从分枝杆菌制备细胞壁组分。 em> 101:91-107。
  3. Bhatt,A.,Fujiwara,N.,Bhatt,K.,Gurcha,SS,Kremer,L.,Chen,B.,Chan,J.,Porcelli,SA,Kobayashi,K.,Besra,GSand Jacobs,WR ,Jr.(2007)。 在结核分枝杆菌中缺失kasB会导致耐酸性和亚临床丧失在免疫活性小鼠中的潜伏性结核。美国国家科学院院报(American Proc Natl Acad Sci)美国 104(12):5157-5162。
  4. Hartkoorn,RC,Sala,C.,Neres,J.,Pojer,F.,Magnet,S.,Mukherjee,R.,Uplekar,S.,Boy-Rottger,S.,Altmann,KHand Cole, )。 迈向新的结核病药物:吡哆霉素 - 自然异烟肼 EMBO Mol Med 4(10):1032-1042。
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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. Dupont, C. M. and Kremer, L. (2014). Extraction and Purification of Mycobacterial Mycolic Acids . Bio-protocol 4(20): e1265. DOI: 10.21769/BioProtoc.1265.
  2. Alahari, A., Trivelli, X., Guérardel, Y., Dover, L. G., Besra, G. S., Sacchettini, J. C., Reynolds, R. C., Coxon, G. D. and Kremer, L. (2007). Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria. PLoS One 2(12): e1343.
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