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Chase Assay of Protein Stability in Haloferax volcanii
沃氏嗜盐富饶菌中蛋白质稳定性的追踪测定   

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Abstract

Highly regulated and targeted protein degradation plays a fundamental role in almost all cellular processes. Determination of the protein half-life by the chase assay serves as a powerful and popular strategy to compare the protein stability and study proteolysis pathways in cells. Here, we describe a chase assay in Haloferax volcanii, a halophilic archaeon as the model organism.

Keywords: Archaea(古生菌), Ubiquitin-proteasome system(泛素 - 蛋白酶体系统), Targeted proteolysis(靶向蛋白水解), SAMP(SAMP), Chase assay(追踪测定)

Background

In eukaryotes, the ubiquitin proteasome system plays a major role in highly selective and targeted proteolysis (Glickman and Ciechanover, 2002). Recent evidence shows that small archaeal ubiquitin-like modifier proteins or SAMPs also function in targeting proteins for destruction by proteasomes (Maupin-Furlow, 2014; Anjum et al., 2015; Fu et al., 2016). Measurement of the protein half-life in vivo provides a direct way to study the proteolysis pathway. Cycloheximide chase and pulse-chase assays are commonly used to monitor the degradation of the targeted protein in eukaryotes (Zhou, 2004). The former method is utilized to determine the half-life of all cellular proteins after inhibition of translation elongation by cycloheximide; whereas, the pulse-chase assay measures the turnover of newly synthesized (pulse-labeled) proteins without interfering the normal cell growth. Compared with the eukaryotic system, a rapid and simple method to determine the half-life of a given protein in archaea is not well established. Therefore, we developed a protocol to measure the protein stability in vivo for the salt-loving archaeon Haloferax volcanii. Inhibitors of translation (anisomycin) and transcription (actinomycin D) are utilized to minimize the synthesis of new protein in this archaeon. TBP2, a TATA-binding protein (TBP) modified by ubiquitin-like isopeptide bonds in Haloferax volcanii, serves as the model protein substrate in this study.

Materials and Reagents

  1. Sterilized wooden stick
  2. Parafilm
  3. Zip-lock plastic bags
  4. 13 x 100 mm2 culture tubes (Fisher Scientific, catalog number: 14-961-27 )
  5. 1.5 ml microcentrifuge tube (Fisher Scientific, catalog number: 02-681-320 )
  6. Polyvinylidene difluoride (PVDF) membrane (GE Healthcare, catalog number: 10600023 )
  7. X-ray film (RPI, catalog number: 248300 )
  8. 2.0 ml microcentrifuge tube (Fisher Scientific, catalog number: 02-681-321 )
  9. Disposable plastic cuvettes (Fisher Scientific, catalog number: 149-551-27 )
  10. Gloves (Fisher Scientific, catalog number: 19-130-1597C )
  11. Rack LTS tips
    ‘P20’ 2-20 µl (Mettler-Toledo, Rainin, catalog number: 17001865 )
    ‘P200’ 20-200 µl (Mettler-Toledo, Rainin, catalog number: 17001863 )
    ‘P1000’ 100-1,000 µl (Mettler-Toledo, Rainin, catalog number: 17001864 )
  12. Sterile polystyrene disposable serological 10 ml pipets with magnifier stripe (Fisher Scientific, catalog number: 13-678-11E )
  13. Nalgene rapid-flow sterile disposable bottle top 0.2 µm filters with surfactant-free cellulose acetate (SFCA) membrane (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 290-3320 )
  14. Haloferax volcanii cells (parent and ubiquitin-like proteasome system mutants) carrying the reporter plasmid pJAM2201 encoding Flag-SAMP2 and TBP2-StrepII under control of the P2rrnA constitutive promoter. Plasmid pJAM202c served as the empty vector control. Please refer to strain and plasmid information published as Table S2 in (Fu et al., 2016)
  15. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  16. Agar (Sigma-Aldrich, catalog number: A7002 )
  17. Novobiocin (Sigma-Aldrich, catalog number: N1628 )
  18. Actinomycin D (Sigma-Aldrich, catalog number: A1410 )
  19. Anisomycin (Sigma-Aldrich, catalog number: A9789 )
  20. Acetone (Sigma-Aldrich, catalog number: 650501 )
  21. Methanol (Fisher Scientific, catalog number: A413 )
  22. CDP-Star (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: T2304 )
  23. Coomassie brilliant blue R-250 staining solution (Bio-Rad Laboratories, catalog number: 1610436 )
  24. Antibodies
    1. Anti-StrepII polyclonal antibody (in mouse) (QIAGEN, catalog number: 34850 )
    2. Goat anti-mouse IgG (whole molecule)-alkaline phosphatase-linked antibody (Sigma-Aldrich, catalog number: A5153 )
    3. Alkaline phosphatase-linked anti-Flag M2 monoclonal antibody (Sigma-Aldrich, catalog number: A9469 )
  25. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S642-12 )
  26. Magnesium chloride hexahydrate (MgCl2·6H2O) (Fisher Scientific, catalog number: M35-12 )
  27. Potassium sulfate (K2SO4) (Fisher Scientific, catalog number: P304-3 )
  28. Calcium chloride dihydrate (CaCl2·2H2O) (Fisher Scientific, catalog number: C79-500 )
  29. Tryptone (BD, BactoTM, catalog number: 211705 )
  30. Yeast extract (BD, BBL, catalog number: 211929 )
  31. Deionized H2O
  32. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: BP359-212 )
  33. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Fisher Scientific, catalog number: M63-3 )
  34. Potassium chloride (KCl) (Fisher Scientific, catalog number: P217-3 )
  35. Tris-base (Fisher Scientific, catalog number: BP152-1 )
  36. Sodium dodecyl sulfate (SDS) (Fisher Scientific, catalog number: BP166-500 )
  37. Glycine (Bio-Rad Laboratories, catalog number: 1610718 )
  38. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  39. Bromopheno blue (Sigma-Aldrich, catalog number: B5525 )
  40. Acrylamide (30%) (Fisher Scientific, catalog number: EC890450ML )
  41. ATCC974 medium (see Recipes)
  42. Concentrated salt water (SW) stock solution at 30% (w/v) (see Recipes)
  43. 2x SDS reducing buffer (see Recipes)

Equipment

  1. Incubator and shaker (42 °C) (Eppendorf, New Brunswick Scientific)
  2. SmartSpec Plus spectrophotometer (Bio-Rad Laboratories, catalog number: 1702525 )
    Note: This product has been discontinued.
  3. Centrifuge (Eppendorf, model: 5418 )
  4. Basic power supply (Bio-Rad Laboratories, catalog number: 1645050 )
  5. Refrigerator (4 °C) (Frigidare)
  6. Ultra-low temperature freezer (-80 °C) (Eppendorf, New BrunswickTM, model: C660-86 )
  7. Vortex mixer (Thermolyne, model: 37600 )
  8. Chemical fume hood (Siemens, catalog number: 537-473 )
  9. Pipettes (2-20 µl, 20-200 µl, 100-1,000 µl) (Rainin type LTS)
  10. pH meter (Corning, model: 320 )
  11. Scanner (Epson, model: 3170 Photo )
  12. Autoclave (Consolidated Sterilizer Systems, model: SR-24C-PB )
  13. Mini trans-blot module (Bio-Rad Laboratories, catalog number: 1703935EDU )
  14. Gel electrophoresis chamber (Bio-Rad Laboratories, catalog number: 1658005 )
  15. Konica X-ray film processor (Konica Minolta, model: QX60A )
  16. Electrophoresis systems autoradiography cassette (Fisher Scientific, FisherBiotech, model: FBXC810 )
  17. Siemens Vantage Reverse Osmosis Systems (M21 series, Siemens, model: M21R004EA ) with EVOQUA filters (Siemens, model: C1207098 ) and Atlantic Ultraviolet Germicidal UV Equipment (Siemens, model: MP49 ) (water purification system used for generating deionized water)

Software

  1. ImageJ (imagej.net/Particle_Analysis)
  2. Microsoft Excel (Microsoft Office 365 ProPlus)

Procedure

  1. Grow Haloferax volcanii cells to stationary phase (OD600 2.0-3.0) in ATCC974 medium and sterilely mix 1:1 with 40% (v/v) glycerol. Store the 20% (v/v) glycerol stock of cells at -80 °C (long-term).
  2. Inoculate the cells by taking a tip of the 20% (v/v) glycerol stock using a sterilized wooden stick and streaking for isolation onto a culture plate that has ATCC974 (described in Recipes section) medium supplemented with 2% (w/v) agar for solid medium and novobiocin (0.2 μg ml-1) to maintain the plasmid DNA. Wrap the plates with Parafilm and transfer to zip-lock plastic bags to prevent loss of moisture.
  3. Incubate the cells for around 100 h at 42 °C. Store the cells on the plates in the sealed bags at room temperature in the dark for up to 2 weeks.
  4. Inoculate isolated colonies of the Haloferax volcanii strains carrying plasmid pJAM2201 into 4 ml ATCC974 medium with novobiocin (0.2 μg ml-1). Grow the cells at 42 °C in liquid medium with rotary shaking at 200 rpm in 13 x 100 mm2 culture tubes.
  5. Monitor the growth of the cells at OD600 using the spectrophotomer. After around 20 h of incubation, the cells will reach log-phase (OD600 of 0.4-0.7). Normalize the log-phase cells by subculturing the cells to an OD600 of 0.06 in 5.1 ml of fresh ATCC974 medium (the added 0.1 ml of medium compensates for loss of culture volume during growth due to evaporation). Grow the cells in the liquid medium for 24 h to again reach log phase with rotary shaking at 200 rpm in 13 x 100 mm2 culture tubes at 42 °C.
  6. Determine the OD600 of the log-phase cells by using 0.2 ml of the cell culture and the spectrophotometer.
  7. Harvest the remaining cells by centrifugation (10,000 x g, 6 min, 25 °C) and resuspend the cell pellets by vortexing for 30 sec in 4.2 ml pre-warmed concentrated salt water solution (described in Recipes section) to a final OD600 of 0.7.
  8. Collect 1 ml of the cells as the 0 min sample. Treat the remaining cells with 20 μg ml-1 actinomycin D and 50 μg ml-1 anisomycin. Actinomycin D is added into the culture right after the addition of anisomycin. Grow cells with rotary shaking at 200 rpm in 13 x 100 mm2 culture tubes at 42 °C.
    Note: Actinomycin D is dissolved in acetone to make a stock solution at 10 mg ml-1. Anisomycin is dissolved in methanol to make a stock solution at 20 mg ml-1. Actinomycin D and anisomycin tend to form some precipitants in high-salt buffer, vortex the solution to fully dissolve the antibiotics.
  9. Transfer 1 ml of antibiotic-treated cells from the culture tube to a 1.5 ml microcentrifuge tube at various intervals (20, 40, 60 min). Immediately harvest the cells at each interval by centrifugation (10,000 x g, 6 min, 4 °C). Carefully remove the supernatant by pipetting as shown in Figure 1 without touching the cell pellets. Freeze the cell pellets at -80 °C before lysis.
    Note: Vortex the solution in the culture tube for 10 sec right before transferring from the culture tube. The cell pellets are frozen at -80 °C for 1-3 h before further processing of the samples.


    Figure 1. Image showing how to remove the supernatant without touching the cell pellets. Haloferax volcanii cells after centrifugation appear as red pellets. The authors recommend to put the small tip on top of the big tip when using a 1,000 μl pipette for pipetting.

  10. Resuspend the harvested cells by up and down pipetting in SDS reducing buffer (described in the Recipes section). In this experiment, 1 ml cell culture is collected and ~0.07 OD600 units of cells are analyzed in 5 µl of sample by SDS-PAGE. Thus, the volume of the SDS reducing buffer is added at 71.4 (μl) times the OD600 of the cell pellet. Vol (SDS reducing buffer) (μl) = OD600 x Vol (harvested cell culture) (ml) x Vol (sample loaded on gel) (μl)/0.07.
  11. Boil the cell suspension 3 times (5 min each time with 30 sec vortex between each time).
  12. Analyze the sample (5 µl per lane) by SDS-PAGE and Western blotting following a regular protocol. Generally, proteins are separated by 12% SDS-PAGE and electroblotted onto PVDF membranes by wet transfer. Equivalent protein loading is determined by OD600 of cell culture (0.08 OD600 units per lane) and confirmed by staining parallel gels with Coomassie blue. Tbp2-StrepII is detected by mouse anti-StrepII polyclonal antibody (1:5,000 dilution) followed by goat anti-mouse IgG (whole molecule)-alkaline phosphatase-linked antibody (1:10,000 dilution). Flag-SAMP is detected by alkaline phosphatase-linked anti-Flag M2 monoclonal antibody (1:10,000 dilution). Targeted proteins on PVDF are visualized by chemiluminescence using CDP-Star with X-ray film according to the user guidelines.
  13. The X-ray films are scanned and the intensity of protein bands by Western blotting is quantified using ImageJ software.

Data analysis

Scan the region of X-ray film containing protein bands to produce a TIF file. Quantification of protein bands on the image is achieved by ImageJ software according to the user guidelines. Generally, purified TBP2 is used as the standard and a range of 0.5 ng to 50 ng protein serves as a range for the standard curve. Each TBP2 protein band is included in the same rectangle and the signal intensity reflected by the size of a peak is plotted. Details about quantification of protein by using ImageJ software can refer to (http://www.openwetware.org/wiki/Protein_Quantification_Using_ImageJ). Results are expressed as the percent change from time zero, which is set at 1.00. For example, measurement of the stability of TBP-StrepII and Flag-SAMP2 in the wild-type Haloferax volcanii strain by this chase assay has been published in mBio (Fu et al., 2016). Figure 2 for the representative data is originally published as Figure S2 by Fu et al. (2016).


Figure 2. TBP2 degradation in wild-type strain. Chase assays were performed in the wild-type strain expressing Flag-SAMP2 and TBP2-StrepII. Log-phase cells were treated with 20 µg ml-1 actinomycin D and 50 µg ml-1 anisomycin for the indicated times and collected. TBP2 and SAMP2 protein levels were determined by anti-StrepII antibody and anti-Flag antibody, respectively. Equal loading was confirmed by CB staining. Experiments were performed in at least biological duplicates, and representative images are shown. ¥, coexpressed in trans. The dataset used in this figure was originally published in Fu et al. (2016).

Notes

Please note that the measure of TBP2 stability in this study was conducted when cells were grown in the standard culture condition. Knowing the environmental factors that trigger the turnover of targeted proteins and conducting the experiment in conditions where the targeted proteins are degraded will be highly recommended when other protein substrates are tested by this chase assay.

Recipes

  1. ATCC974 medium
    1. Dissolve 125 g NaCl, 50 g MgCl2·6H2O, 5 g K2SO4, 0.132 g CaCl2·2H2O, 5 g tryptone, and 5 g yeast extract in 750 ml deionized H2O. The medium will appear as a clear solution when all the chemicals are fully dissolved
    2. Adjust the pH of the solution to 6.8 by adding 0.5 N NaOH drop by drop
    3. Adjust the volume of the solution to 1 L final. Transfer the solution to 2 x 1 L glass media bottles (500 ml medium per bottle) and autoclave on the liquid cycle for 25 min
    4. Store medium at room temperature for up to 6 months.
    5. For growth of Haloferax volcanii strains carrying plasmid pJAM2201 (and the empty vector control pJAM202c), the ATCC974 medium is supplemented with novobiocin to a final concentration of 0.2 μg ml-1. The medium is cooled to room temperature prior to addition of the antibiotic and used immediately, as the antibiotic is sensitive to high temperature and light
  2. Concentrated salt water (SW) stock solution at 30% (w/v)
    1. Dissolve 240 g NaCl, 30 g MgCl2·6H2O, 35 g MgSO4·7H2O, and 7 g KCl in 650 ml deionized H2O
    2. Add 10 ml Tris-Cl (1 M, pH 7.5) to the solution to adjust the pH to 7.4
    3. Transfer the pH adjusted solution to a large, graduated cylinder, then top up with deionized H2O to a final volume of 1 L
    4. Filter-sterilize the solution using a sterile disposable 0.2 µm filter.
    5. Store the SW stock for up to 6 months at room temperature
  3. 2x SDS reducing buffer
    100 mM Tris-Cl buffer at pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 0.6 mg ml-1 bromophenol blue, and 5% (v/v) β-mercaptoethanol

Acknowledgments

This work was funded by US Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences, Physical Biosciences Program (DE-FG02-05ER15650), USDA National Institute of Food and Agriculture (Hatch 1005900), and NIH | National Institute of General Medical Sciences (NIGMS) (NIH R01 GM57498-15).

References

  1. Anjum, R. S., Bray, S. M., Blackwood, J. K., Kilkenny, M. L., Coelho, M. A., Foster, B. M., Li, S., Howard, J. A., Pellegrini, L., Albers, S. V., Deery, M. J. and Robinson, N. P. (2015). Involvement of a eukaryotic-like ubiquitin-related modifier in the proteasome pathway of the archaeon Sulfolobus acidocaldarius. Nat Commun 6: 8163.
  2. Fu, X., Liu, R., Sanchez, I., Silva-Sanchez, C., Hepowit, N. L., Cao, S., Chen, S. and Maupin-Furlow, J. (2016). Ubiquitin-like proteasome system represents a eukaryotic-like pathway for targeted proteolysis in archaea. MBio 7(3).
  3. Glickman, M. H. and Ciechanover, A. (2002). The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82(2): 373-428.
  4. Maupin-Furlow, J. A. (2014). Prokaryotic ubiquitin-like protein modification. Annu Rev Microbiol 68: 155-175.
  5. Zhou, P. (2004). Determining protein half-lives. Methods Mol Biol 284: 67-77.

简介

高度调节和靶向的蛋白质降解在几乎所有的细胞过程中发挥重要作用。通过追踪测定法测定蛋白质半衰期是比较蛋白质稳定性和研究细胞中蛋白水解途径的有力和普遍的策略。在这里,我们描述了作为模型生物体的嗜盐古细菌Haloferax volcanii 中的追踪测定。

背景 在真核生物中,泛素蛋白酶体系统在高选择性和靶向性蛋白水解中起主要作用(Glickman and Ciechanover,2002)。最近的证据表明,小型古细菌泛素样修饰蛋白或SAMPs也起到了蛋白酶体破坏的靶向蛋白的作用(Maupin-Furlow,2014; Anjum等人,2015; Fu et al。 。,2016)。体内蛋白质半衰期的测量提供了研究蛋白水解途径的直接途径。环己酰胺追踪和脉冲追踪测定通常用于监测真核生物中靶向蛋白质的降解(Zhou,2004)。前一种方法用于确定放线菌酮抑制平移伸长后所有细胞蛋白的半衰期;而脉冲追踪测定法测量新合成(脉冲标记)蛋白质的周转率,而不干扰正常的细胞生长。与真核系统相比,确定古细菌中一种给定蛋白质半衰期的快速简便方法尚未明确。因此,我们开发了一种方案,用于测量盐爱好的考古Haloferax volcanii的体内蛋白质稳定性。使用翻译抑制剂(茴香霉素)和转录(放线菌素D)来最小化该古细菌中新蛋白质的合成。 TBP2是在Haloferax volcanii中通过泛素样异肽键修饰的TATA结合蛋白(TBP),在本研究中用作模型蛋白质底物。

关键字:古生菌, 泛素 - 蛋白酶体系统, 靶向蛋白水解, SAMP, 追踪测定

材料和试剂

  1. 灭菌木棍
  2. 石蜡膜
  3. 拉链锁胶袋
  4. 13×100mm 培养管(Fisher Scientific,目录号:14-961-27)
  5. 1.5ml微量离心管(Fisher Scientific,目录号:02-681-320)
  6. 聚偏二氟乙烯(PVDF)膜(GE Healthcare,目录号:10600023)
  7. X光胶片(RPI,目录号:248300)
  8. 2.0ml微量离心管(Fisher Scientific,目录号:02-681-321)
  9. 一次性塑料比色皿(Fisher Scientific,目录号:149-551-27)
  10. 手套(Fisher Scientific,目录号:19-130-1597C)
  11. 机架LTS提示
    'P20'2-20μl(Mettler-Toledo,Rainin,目录号:17001865)
    'P200'20-200μl(Mettler-Toledo,Rainin,目录号:17001863)
    'P1000'100-1,000μl(Mettler-Toledo,Rainin,目录号:17001864)
  12. 无菌聚苯乙烯一次性血清学10ml带放大镜条的移液管(Fisher Scientific,目录号:13-678-11E)
  13. 具有无表面活性剂的醋酸纤维素(SFCA)膜(Thermo Fisher Scientific,Thermo Scientific TM,目录号:290-3320)的Nalgene快速无菌一次性瓶顶部0.2μm过滤器
  14. 携带编码Flag-SAMP2和TBP2-StrepII的报告质粒pJAM2201的Haloferax volcanii细胞(亲本和泛素样蛋白酶体系突变体)在P2 /sub>组成型启动子。质粒pJAM202c作为空载体对照。请参阅(Fu等人,2016)表S2所示的菌株和质粒信息。
  15. 甘油(Sigma-Aldrich,目录号:G5516)
  16. 琼脂(Sigma-Aldrich,目录号:A7002)
  17. 新生霉素(Sigma-Aldrich,目录号:N1628)
  18. 放线菌素D(Sigma-Aldrich,目录号:A1410)
  19. 异烟霉素(Sigma-Aldrich,目录号:A9789)
  20. 丙酮(Sigma-Aldrich,目录号:650501)
  21. 甲醇(Fisher Scientific,目录号:A413)
  22. (Thermo Fisher Scientific,Molecular Probes TM ,目录号:T2304)
  23. 考马斯亮蓝R-250染色溶液(Bio-Rad Laboratories,目录号:1610436)
  24. 抗体
    1. 抗StrepII多克隆抗体(小鼠)(QIAGEN,目录号:34850)
    2. 山羊抗小鼠IgG(全分子) - 碱性磷酸酶连接的抗体(Sigma-Aldrich,目录号:A5153)
    3. 碱性磷酸酶连接的抗Flag M2单克隆抗体(Sigma-Aldrich,目录号:A9469)
  25. 氯化钠(NaCl)(Fisher Scientific,目录号:S642-12)
  26. 氯化镁六水合物(MgCl 2·6H 2 O)(Fisher Scientific,目录号:M35-12)
  27. 硫酸钾(K 2 O 3 SO 4)(Fisher Scientific,目录号:P304-3)
  28. 氯化钙二水合物(CaCl 2·2H 2 O)(Fisher Scientific,目录号:C79-500)
  29. Tryptone(BD,Bacto TM ,目录号:211705)
  30. 酵母提取物(BD,BBL,目录号:211929)
  31. 去离子H 2 O O
  32. 氢氧化钠(NaOH)(Fisher Scientific,目录号:BP359-212)
  33. 硫酸镁七水合物(MgSO 4·7H 2 O)(Fisher Scientific,目录号:M63-3)
  34. 氯化钾(KCl)(Fisher Scientific,目录号:P217-3)
  35. Tris-base(Fisher Scientific,目录号:BP152-1)
  36. 十二烷基硫酸钠(SDS)(Fisher Scientific,目录号:BP166-500)
  37. 甘氨酸(Bio-Rad Laboratories,目录号:1610718)
  38. β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  39. 溴苯酚蓝(Sigma-Aldrich,目录号:B5525)
  40. 丙烯酰胺(30%)(Fisher Scientific,目录号:EC890450ML)
  41. ATCC974培养基(参见食谱)
  42. 30%(w/v)的浓盐水(SW)储备溶液(参见食谱)
  43. 2x SDS减缓缓冲液(见配方)

设备

  1. 孵化器和振荡器(42°C)(Eppendorf,New Brunswick Scientific)
  2. SmartSpec Plus分光光度计(Bio-Rad Laboratories,目录号:1702525)
    注意:本产品已停产。
  3. 离心机(Eppendorf,型号:5418)
  4. 基本电源(Bio-Rad Laboratories,目录号:1645050)
  5. 冰箱(4°C)(Frigidare)
  6. 超低温冷冻机(-80℃)(Eppendorf,New Brunswick TM,型号:C660-86)
  7. 涡旋搅拌机(Thermolyne,型号:37600)
  8. 化学通风橱(西门子,目录号:537-473)
  9. 移液管(2-20μl,20-200μl,100-1,000μl)(Rainin型LTS)
  10. pH计(康宁,型号:320)
  11. 扫描仪(Epson,型号:3170照片)
  12. 高压釜(综合灭菌系统,型号:SR-24C-PB)
  13. 微型印迹模块(Bio-Rad Laboratories,目录号:1703935EDU)
  14. 凝胶电泳室(Bio-Rad Laboratories,目录号:1658005)
  15. 柯尼卡X光片处理器(柯尼卡美能达,型号:QX60A)
  16. 电泳系统放射自显影盒(Fisher Scientific,FisherBiotech,型号:FBXC810)
  17. 西门子Vantage反渗透系统(M21系列,西门子,型号:M21R004EA)与EVOQUA过滤器(西门子,型号:C1207098)和大西洋紫外线杀菌设备(西门子,型号:MP49)(用于产生去离子水的水净化系统) />

软件

  1. ImageJ(imagej.net/Particle_Analysis)
  2. Microsoft Excel(Microsoft Office 365 ProPlus)

程序

  1. 在ATCC974培养基中将Haloferax volcanii细胞生长至固定相(OD 600,2.0-3.0),并与40%(v/v)甘油无菌混合1:1。将20%(v/v)的甘油储存液储存在-80°C(长期)
  2. 通过使用灭菌的木棒将20%(v/v)甘油储液的末端接种,并将条带分离到具有ATCC974(描述于食谱部分)的补充有2%(w/v)用于固体培养基和新生霉素(0.2μg/ml)的琼脂以维持质粒DNA。用Parafilm包裹板,并转移到拉链锁塑料袋以防止水分流失。
  3. 在42℃下孵育细胞约100小时。将细胞在室温下在黑暗中储存在密封袋中的板上长达2周。
  4. 将带有质粒pJAM2201的携带 菌株的分离菌落接种到具有新生霉素(0.2μg/ml)的4ml ATCC974培养基中。在液体培养基中以42rpm的速度在200rpm的旋转振荡下在13×100mm的培养管中培养细胞。
  5. 使用分光光度计监测OD 600的细胞生长。孵育约20小时后,细胞将达到对数期(OD 600> 0.4-0.7)。通过在5.1ml新鲜ATCC974培养基中将细胞传代培养至0.06的OD 600,校准对数相细胞(加入0.1ml培养基补偿由于蒸发而在生长期间的培养体积的损失)。在42℃下,在13×100mm 2培养管中,以200rpm旋转振荡,在液体培养基中培养细胞24小时以再次达到对数期。
  6. 通过使用0.2ml细胞培养物和分光光度计来确定对数相细胞的OD 600。
  7. 通过离心(10,000×10 6分钟,25℃)收获剩余的细胞,并通过在4.2ml预热浓缩盐水溶液(在方法部分中描述)中涡旋30秒来重悬细胞沉淀,达到0.7的最终OD 600。
  8. 收集1 ml细胞作为0 min样品。用20μg/ml的放线菌素D和50μg/ml的茴香霉素处理剩余的细胞。放线菌素D在加入茴香霉素后立即加入到培养基中。在42℃下在13×100mm 2培养管中以200rpm旋转振荡培养细胞。
    注意:放线菌素D溶解在丙酮中以制备10毫克/升的储备溶液。将异青霉素溶于甲醇中以制备20毫克/升的储备溶液。放线菌素D和茴香霉素倾向于在高盐缓冲液中形成一些沉淀剂,涡旋溶液以完全溶解抗生素。
  9. 将1ml抗生素处理的细胞从培养管转移到1.5ml微量离心管中,每个间隔(20,40,60分钟)。通过离心(10,000×g,6分钟,4℃)立即在每个间隔收获细胞。如图1所示,通过移液小心地去除上清液,而不接触细胞沉淀。在裂解前将细胞沉淀物冷冻至-80°C。
    注意:在从培养管转移之前,将培养管中的溶液涡旋10秒钟。细胞沉淀在-80℃下冷冻1-3小时,然后进一步处理样品。


    图1.显示如何在不接触细胞沉淀的情况下清除上清液的图像。离心后的卤虫感染细胞以红色颗粒显示。作者建议在使用1,000μl移液管进行移液时,将小尖头放在大尖顶上。

  10. 通过在SDS还原缓冲液中上下移液来重新收获收获的细胞(在食谱部分中描述)。在该实验中,收集1ml细胞培养物,并通过SDS-PAGE在5μl样品中分析〜0.07 OD 600单位的细胞。因此,将SDS还原缓冲液的体积加入细胞沉淀物的OD 600的71.4(μl)倍。 Vol(SDS还原缓冲液)(μl)= OD 600(收获的细胞培养物)(ml)×Vol(在凝胶上加载的样品)(μl)/0.07。
  11. 将细胞悬浮液煮3次(每次5分钟,每次30秒涡旋)。
  12. 按照常规方案,通过SDS-PAGE和Western印迹分析样品(每泳道5μl)。通常,蛋白质通过12%SDS-PAGE分离,并通过湿转移电印迹到PVDF膜上。通过细胞培养物的OD 600(每道0.08 OD 600)确定等效蛋白质负载,并通过用考马斯兰染色平行凝胶来证实。通过小鼠抗StrepII多克隆抗体(1:5000稀释),然后用山羊抗小鼠IgG(全分子) - 碱性磷酸酶连接的抗体(1:10,000稀释)检测Tbp2-StrepII。通过碱性磷酸酶连接的抗Flag M2单克隆抗体(1:10,000稀释)检测Flag-SAMP。根据用户指南,使用CDP-Star与X射线胶片通过化学发光显示PVDF上的靶向蛋白质。
  13. 扫描X射线胶片,Western印迹法测定蛋白质条带的强度,并用ImageJ软件进行定量

数据分析

扫描含有蛋白质条带的X射线胶片区域以产生TIF文件。图像上的蛋白质带的定量通过ImageJ软件根据用户指南实现。通常,使用纯化的TBP2作为标准品,0.5ng至50ng蛋白质的范围作为标准曲线的范围。每个TBP2蛋白质带都包含在同一个矩形中,并绘制了峰值大小反映的信号强度。使用ImageJ软件量化蛋白质的细节可参考( http://www .openwetware.org/wiki/Protein_Quantification_Using_ImageJ )。结果表示为从零开始的百分比变化,设置为1.00。例如,通过该追踪测定法测定野生型Haloferax volcanii 菌株中TBP-StrepII和Flag-SAMP2的稳定性已经在mBio(Fu >等,,2016)。代表性数据的图2最初由Fu等人发表如图S2所示。 (2016)。


图2.野生型菌株中的TBP2降解。在表达Flag-SAMP2和TBP2-StrepII的野生型菌株中进行Chase测定。对照期细胞用20μg/ml的放线菌素D和50μg/ml的茴香霉素处理指定的时间并收集。分别通过抗StrepII抗体和抗Flag抗体测定TBP2和SAMP2蛋白水平。通过CB染色确认相等载量。至少在生物重复中进行实验,并显示代表性的图像。 ¥,coexpressed in trans 。该图中使用的数据集最初出版于Fu等人。 (2016年)。

笔记

请注意,当细胞在标准培养条件下生长时,本研究中测量TBP2稳定性。了解引发目标蛋白质周转的环境因素,并在靶向蛋白质降解的条件下进行实验,将会强烈推荐其它蛋白质底物通过该追踪测定法进行测试。

食谱

  1. ATCC974培养基
    1. 溶解125g NaCl,50g MgCl 2·6H 2 O,5g K 2 SO 4,0.132 g CaCl 2·2H 2 O,5g胰蛋白胨和5g酵母提取物在750ml去离子H 2 O中的溶液中。当所有化学品完全溶解时,介质将显示为明确的解决方案
    2. 通过加入0.5N NaOH逐滴将溶液的pH调节至6.8
    3. 将解决方案的体积调整为1 L最终。将溶液转移到2×1L玻璃介质瓶(每瓶500ml)中,并在液体循环上高压灭菌25分钟
    4. 储存介质在室温下长达6个月。
    5. 对于携带质粒pJAM2201(和空载体对照pJAM202c)的Haloferax volcanii菌株的生长,将ATCC974培养基补充有新生霉素至终浓度为0.2μg/ml。培养基在加入抗生素前冷却至室温,立即使用,因为抗生素对高温和轻度敏感
  2. 浓度为30%(w/v)的盐水(SW)储备溶液
    1. 溶解240g NaCl,30g MgCl 2·6H 2 O,35g MgSO 4·7H 2 O ,和7g KCl在650ml去离子H 2 O中的溶液
    2. 向溶液中加入10ml Tris-Cl(1M,pH 7.5)将pH调节至7.4
    3. 将pH调节的溶液转移到一个大的量筒上,然后用去离子H 2 O加满至1L的最终体积
    4. 使用无菌一次性0.2μm过滤器对溶液进行过滤消毒。
    5. 在室温下存放SW库存长达6个月
  3. 2x SDS减缓缓冲液
    pH 6.8的100mM Tris-Cl缓冲液,4%(w/v)SDS,20%(v/v)甘油,0.6mg ml-1溴酚蓝和5%(v/v)β-巯基乙醇, >

致谢

这项工作由美国能源部,基础能源科学办公室,化学科学,地球科学和生物科学部,物理生物科学计划(DE-FG02-05ER15650),美国农业部国家食品和农业研究所(Hatch 1005900)和NIH |国立综合医学研究所(NIGMS)(NIH R01 GM57498-15)。

参考文献

  1. Anjum,RS,Bray,SM,Blackwood,JK,Kilkenny,ML,Coelho,MA,Foster,BM,Li,S.,Howard,JA,Pellegrini,L.,Albers,SV,Deery,MJ and Robinson,NP 2015)。涉及真核样泛素相关修复剂在古细菌Sulfolobus acidocaldarius 的蛋白酶体途径中。 6:8163。
  2. Fu,X.,Liu,R.,Sanchez,I.,Silva-Sanchez,C.,Hepowit,NL,Cao,S.,Chen,S.and Maupin-Furlow,J。(2016)。 MBio 7(3)。
  3. Glickman,MH和Ciechanover,A。(2002)。泛素 - 蛋白酶体蛋白酶解途径:为了构建起到破坏作用。 82(2):373-428。
  4. Maupin-Furlow,JA(2014)。  原核泛素 - 如蛋白质修饰。 Annu Rev Microbiol 68:155-175。
  5. Zhou,P.(2004)。  确定蛋白质半衰期,生活。方法Mol Biol 284:67-77。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Fu, X. and Maupin-Furlow, J. A. (2017). Chase Assay of Protein Stability in Haloferax volcanii. Bio-protocol 7(6): e2186. DOI: 10.21769/BioProtoc.2186.
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