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Isolation of Ribosomal Particles from the Unicellular Cyanobacterium Synechocystis sp. PCC 6803
从单细胞蓝细菌集胞藻PCC 6803 中分离核糖体颗粒   

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Abstract

Isolation of ribosomal particles is an essential step in the study of ribosomal components as well as in the analysis of trans-acting factors that interact with the ribosome to regulate protein synthesis and modulate the expression profile of the cell in response to different environmental conditions. In this protocol, we describe a procedure for the isolation of 70S ribosomes from the unicellular cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We have successfully used this protocol in our study of the cyanobacterial ribosomal-associated protein LrtA, which is a homologue of bacterial HPF (hibernation promoting factor) (Galmozzi et al., 2016).

Keywords: Ribosome(核糖体), Sucrose gradients(蔗糖梯度), Synechocystis(集胞藻), Cyanobacteria(蓝细菌), Photosynthetic prokaryotes(光合原核生物)

Background

Few biochemical studies have been reported for cyanobacterial ribosomes. Anabaena variabilis strain M3 (PCC 7118, ATCC 27892) 70S ribosomal particles have been isolated by differential centrifugation and then, ribosomal proteins were analysed by two-dimensional electrophoresis (Sato et al., 1998). Ribosomes have also been prepared from Synechococcus sp. PCC 6301 cells using a protocol combining differential centrifugation and sucrose step gradients (Sugita et al., 2000). Fractionation of cell extracts by differential centrifugation has also been employed in the preparation of ribosomal samples for the development of an in vitro translation system in different Synechococcus strains (Mutsuda and Sugiura, 2006). The method described here for Synechocystis, based on the one described for Synechococcus (Sugita et al., 2000), allows purification of ribosomal particles using ultracentrifugation of linear sucrose gradients.

Materials and Reagents

  1. Centrifuge tubes, polypropylene 15 ml (Beckman Coulter, catalog number: 342082 )
  2. 1.5 ml microcentrifuge tubes (Microtubes) (Corning, Axygen®, catalog number: MCT-150-C )
  3. 1.5 ml polystyrene spectrophotometer cuvettes (KARTELL SPA CIA DELLE INDUSTRIE, catalog number: 1938 )
  4. Ultracentrifuge thin-wall polyallomer tubes 13.2 ml (Beckman Coulter, catalog number: 331372 )
  5. 0.45 μm Millipore filter (MILLEX-HV PVDF) (EMD Millipore, catalog number: SLHV033RS )
  6. Alumina type A-5 (Sigma-Aldrich, catalog number: A2039 )
    Note: This product has been discontinued.
  7. Micropipette tips (200 μl) (Daslab, catalog number: 162001X )
  8. Micropipette tips (1,000 μl) (Daslab, catalog number: 162222X )
  9. Silicone tubing (VWR, catalog number: 228-0713 )
  10. Synechocystis sp. PCC 6803 cells (Pasteur Culture Collection of Cyanobacteria)
  11. Milli-Q water
  12. Distilled water
  13. Sodium nitrate (NaNO3) (AppliChem, catalog number: 141702 )
  14. Sodium bicarbonate (NaHCO3) (AppliChem, catalog number: 141638 )
  15. Potassium phosphate dibasic (K2HPO4) (AppliChem, catalog number: 141512 )
  16. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Duchefa Biochemie, catalog number: M0513 )
  17. Citric acid monohydrate (C6H8O7·H2O) (EMD Millipore, catalog number: 100244 )
  18. EDTA-Na2 (Duchefa Biochemie, catalog number: E0511 )
  19. Calcium chloride dihydrate (CaCl2·2H2O) (Merck, catalog number: 2382 )
  20. Citrate Fe-NH4 (Sigma-Aldrich, catalog number: F5879 )
  21. Sodium phosphate (Na2CO3) (Merck, catalog number: 6392 )
  22. Boric acid (H3BO3) (EMD Millipore, catalog number: 100165 )
  23. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (EMD Millipore, catalog number: 1059270 )
  24. Sodium molybdate dehydrate (Na2MoO4·2H2O) (Merck, catalog number: 6521 )
  25. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Merck, catalog number: 8883 )
  26. Cobalt nitrate hexahydrate (Co(NO3)2·6H2O) (AppliChem, catalog number: 131258 )
  27. Copper (II) sulfate pentahydrate (CuSO4·5H2O) (EMD Millipore, catalog number: 102790 )
  28. Tris ultrapure (Duchefa Biochemie, catalog number: T1501 )
  29. Ammonium chloride (NH4Cl) (EMD Millipore, catalog number: 101145 )
  30. Magnesium acetate (Sigma-Aldrich, catalog number: M5661 )
  31. β-mercaptoethanol (EMD Millipore, catalog number: 805740 )
  32. Glycerol (AppliChem, catalog number: 131339 )
  33. Liquid nitrogen
  34. Trichloracetic acid (TCA) (EMD Millipore, catalog number: 100807 ) (only for Western)
  35. Acetone (EMD Millipore, catalog number: 100014 ) (only for Western)
  36. Ammonium chloride (EMD Millipore, catalog number: 101145 )
  37. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  38. Hydrochloric acid fuming 47% (EMD Millipore, catalog number: 100317 )
  39. Methanol extra pure (Scharlau, catalog number: ME0301005P )
  40. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A4503 )
  41. Protein assay dye reagent concentrate (Bio-Rad Laboratories, catalog number: 5000006 )
  42. BG11 growth medium (see Recipes)
  43. Extraction buffer (see Recipes)
  44. Sucrose buffer 1 (see Recipes)
  45. Sucrose buffer 2 (see Recipes)
  46. 60% (w/v) sucrose (see Recipes)
  47. 100% (w/v) TCA solution (see Recipes)
  48. 80% (v/v) glycerol (see Recipes)

Equipment

  1. Fluorescent lamps
  2. Erlenmeyer flask 250 ml (Duran Group, catalog number: 21 771 36 )
  3. Flasks and magnetic stirrer for preparation of solutions
  4. Orbital shaker (IKA, model: KS501 )
  5. Roux culture bottle 1 L (Sigma-Aldrich, catalog number: CLS12901L )
  6. Glass bottles 5 L (Iso bottle blue cap 5,000 ml) (Kavalierglass, Simax, catalog number: CFB020 )
  7. Balances (Sartorius, catalog number: TE1502S and Precisa, model: XT120A )
  8. pH meter (HACH LANGE SPAIN, CRISON, model: BASIC 20 )
  9. Flow meters (ABB, model: A6200 )
  10. Compressed air pump
  11. Centrifuge (Beckman Coulter, model: Avanti J-25 )
  12. Rigid polypropylene tube (Fisher Scientific, catalog number: 11723904 )
  13. Fixed angle rotor (Beckman Coulter, model: JLA-16.250 , catalog number: 363934)
  14. Centrifuge bottles polypropylene 250 ml (Beckman Coulter, catalog number: 356011 )
  15. Porcelain mortar and pestle (10 cm Ø) (Fisher Scientific, catalog number: S337621 )
  16. Sterilization oven (JP SELECTA, catalog number: 2000381 )
  17. Autoclave (SANYO Labo Autoclave) (Panasonic Biomedical, model: MLS-3020U )
  18. Automatic micropipettes
  19. Vortex Mixer (Heidolph Instruments, model: Heidolph Reax 2000 )
  20. UV-VIS spectrophotometer (Thermo Electron, model: BioMate 5 )
  21. Fixed angle rotor (Beckman Coulter, model: JA-25.15 )
  22. Gradient maker (Hoefer, catalog number: SG15 )
  23. Peristaltic pump (Pharmacia Biotech, model: Pump P-1 )
  24. Magnetic plate stirrer (Cole-Parmer, catalog number: EW-04807-45 )
  25. Ultracentrifuge (Beckman Coulter, model: XL-80 )
  26. Swinging-bucket rotor (Beckman Coulter, model: SW 41 Ti )
  27. ISCO UA-6 detector UV/VIS (Density Gradient Fractionator, Teledyne Isco, catalog number: 67-9000-176 ) with TRISTM pump (Teledyne Isco)
  28. Microcentrifuge (Eppendorf, model: 5424/5415 R )
  29. Thermoblock (Eppendorf Thermomixer compact) (only for Western)
  30. Metal connectors (Parker-Legris, catalog number: 0180 04 00 )
  31. CO2 cylinder containers

Procedure

An overview of the whole protocol, divided in four steps, is shown in Figure 1.


Figure 1. Scheme of the complete procedure described in the protocol

  1. Cultivation of Synechocystis
    1. Grow Synechocystis cells photoautotrophically at 30 °C in BG11 medium (Rippka et al., 1979) supplemented with 1 g L-1 of NaHCO3 (BG11C) under continuous illumination (50 µmol of photons m-2 sec-1, white light from fluorescent lamps).
      1. Usually cultivation begins with a pre-inoculum (50 ml) growing in Erlenmeyer flasks covered with cotton and placed on an orbital shaker (100 rpm) located in an illuminated room.
      2. These cells are then used to inoculate around 750 ml of BG11C medium in a Roux flask. Finally the culture is scaled to a 5 L bottle (without the cap and covered with cotton).
        Note: To achieve optimal growth, the cultures in Roux flasks and in 5 L bottles are bubbled with CO2-enriched air (1% v/v), using a rigid polypropylene tube, silicone tubing and metal connectors. A compressed air stream drives bubbling and the air-CO2 mixture is established using flow meters.
    2. Estimation of growth phase
      A volume of 3-5 L of cells, in exponential or stationary growth phase depending on the purpose of each experiment, is used. Growth phase is estimated by measuring total chlorophyll content in methanolic extracts of cells (McKinney, 1941).
      1. Centrifuge 1 ml of culture in a 1.5 ml microcentrifuge tube at 12,000 x g.
      2. Discard 950 µl of the supernatant and resuspend the pellet in the remaining 50 µl. Add 950 µl of methanol and vortex vigorously for 1 min.
      3. Centrifuge at 12,000 x g for 2 min. Chlorophyll concentration is calculated by measuring absorbance at 665 nm in the supernatant (E = 74.46 ng-1 ml cm-1).
      4. Cultures with 2-4 µg/ml of total chlorophyll are considered to be in exponential phase and cultures with more than 5 µg/ml are considered stationary.
    3. Harvest cells
      1. Centrifuge at 9,605 x g in a JLA-16.250 Beckman rotor, for 10 min at 4 °C.
      2. Combine the cells in a single bottle and estimate fresh weight. 2-5 g of cells are enough for the procedure.

  2. Breakage and fractionation of Synechocystis cells
    1. Pre-treatment
      All glass materials, porcelain mortar and pestle were heated in a sterilization oven at 200 °C for 3 h and all plastic materials were autoclaved at 121 °C for 20 min.
      Note: RNase-free water was used to prepare all buffers.
    2. Breakage of Synechocystis cells
      Place in a pre-chilled porcelain mortar the same weight of cells and alumina. Put the mortar on top of an ice bucket with ice. Mix with the pestle and once the cells and the alumina are well mixed continue grinding for 2 min. Add around 20 ml extraction buffer (1 or 10 mM Mg-acetate) with β-mercaptoethanol added immediately before use.
    3. Fractionation of Synechocystis cells
      Recover as much material as possible in two polypropylene tubes of 15 ml. Centrifuge at 10,000 x g for 10 min in a JA-25.15 rotor at 4 °C to eliminate the alumina, the cell debris and the unbroken cells. Transfer the supernatant into new 15 ml tubes and centrifuge at 30,000 x g for 30 min in the same rotor at 4 °C. This centrifugation is required to completely eliminate membranes.
    4. Freezing reserve
      Dispense aliquots from the supernatant of this centrifugation step (S30 fraction) in 1.5 ml Eppendorf tubes with 5% (v/v) glycerol. For this process, place the 1.5 ml tubes on ice and add 40 µl of 80% (v/v) glycerol and 600 µl of the S30 fraction to each tube. Mix gently, using a micropipette, and rapidly freeze the tubes in liquid nitrogen. Before freezing reserve an aliquot to determine the total protein amount of this S30 fraction by the standard method of Bradford, using albumin bovine as reference (Bradford et al., 1976). Usually 10-15 µg/µl of total protein concentration is obtained in S30 fraction.
      Notes:
      1. When extraction buffer and gradient solutions contain 1 mM Mg-acetate (low Mg2+), most bacterial ribosomes are dissociated into 30S and 50S subunits but when using 10 mM Mg-acetate (high Mg2+), in the buffers and solutions, the 70S vacant ribosome peak is mainly observed.
      2. It is worth noting that in cyanobacteria, phycobiliproteins represent around 30% of total protein concentration. This fact must be taken into account when considering the amount of the S30 fraction needed for the next step of this procedure.
      3. The S30 fraction can be stored as different aliquots at -80 °C to load separate gradients on different days. This is a backup in case there is any problem in later steps of the protocol, especially in the centrifugation, fractionation or generation of the gradient profiles.

  3. Preparation of sucrose gradients and ultracentrifugation
    1. Preparation of sucrose gradients
      1. Prepare a 10-30% sucrose gradient using a linear gradient maker connected to a peristaltic pump (P1) working at 120 ml/h. Use sucrose buffer 1 and 2 solutions (either with 1 or 10 mM Mg-acetate), with β-mercaptoethanol added immediately before use, and 13.2 ml ultracentrifuge thin-wall polyallomer tubes. Place the gradient maker on top of a magnetic stirrer plate.
      2. Introduce a small magnetic stir bar in the gradient maker compartment connected to the pump. Pour 5.6 ml of sucrose buffer 1 in this compartment and 5.6 ml of sucrose buffer 2 in the other compartment of the gradient maker. The outlet of the peristaltic pump is connected to a glass capillary that is introduced into the gradient tube.
      3. Connect the two compartments of the gradient maker and simultaneously turn on pump P1.
      4. Stop the pump when the solutions introduced in the gradient maker are exhausted, before air bubbles enter the gradient.
      5. Then remove the capillary from the gradient tube slowly so as not to disturb it.
      6. Tubes are stored at 4 °C for about 8 h before use to allow equilibration of the linear sucrose gradients.
      7. Before loading, filter the S30 fraction through a 0.45 µm filter unit.
    2. Ultracentrifugation
      1. Gently layer the S30 fraction (100-300 µl) on top of the 10-30% sucrose gradient using a micropipette and via single drops close to the surface of the sucrose gradient.
      2. Centrifuge at 77,175 x g, for 8 h at 2 °C in a pre-cooled Beckman ultracentrifuge in the SW 41 Ti rotor.

  4. Isolation of ribosomal particles
    1. Carefully remove the gradient tubes from the centrifuge and completely fill each tube with Milli-Q water before placing it in the ISCO fractionation system.
    2. Fractionate the gradient from the top using 60% (w/v) sucrose to push the content of the tube toward the UV detector (A254), with the TRISTM pump (flow rate 1 ml/min). Turn on the recorder (1 cm/min, sensitivity 1) to obtain the sucrose gradient profile while the fractions (0.5 ml) are manually collected.
    3. Place the fractions in a container with liquid nitrogen, as they are collected, to freeze them quickly and then store at -80 °C until use.
      Representative examples of the expected profiles are shown in Figure 2.


      Figure 2. Sedimentation profiles showing the free subunits and 70S vacant ribosomes. The Synechocystis S30 supernatant was fractionated on a 10-30% sucrose density gradient in the presence of 1 mM (A) or 10 mM (B) Mg2+. Small numbers refer to collected fractions. Adapted from (Galmozzi et al., 2016).

    4. The treatment of the fractions from this point depends on the use that will be given to them. In the case of analysis by Western blot, precipitate the total protein with 100% (w/v) TCA (see Recipes).
      1. Add 55 µl of this solution to each fraction (10% TCA final concentration), incubate on ice for 30 min and centrifuge in a microcentrifuge (Eppendorf 5415 R) at maximal speed and 4 °C for 10 min.
      2. Discard the supernatant and add 1 ml of -20 °C chilled acetone to each fraction pellet to eliminate residual TCA.
      3. Remove the acetone carefully as not to affect the integrity of the pellet.
      4. Dry the pellets through evaporation of acetone by incubating the tubes at 60 °C in a thermoblock.
      5. Resuspend the pellets in RNase-free Milli-Q water and add loading buffer for standard electrophoresis protocols.

Notes

Nowadays the analog signal from the ISCO UA-6 detector can be transformed into a digital signal using a Bus-Powered Multifunction DAQ USB Device (NI USB-6008) from National Instruments.

Recipes

  1. BG11 growth medium (Rippka et al., 1979)
    1. BG11C (1 L)
      1.5 g NaNO3
      1 g NaHCO3
      0.2 ml 1 M K2HPO4
      10 ml concentrate BG11 (see below)
      Autoclave at 121 °C, 1 atm for 20 min
    2. Concentrate BG11
      7.5 g/L MgSO4·7H2O
      0.6 g/L citric acid
      0.1 g/L EDTA-Na2
      100 ml A5 solution (see below)
      3.6 g/L CaCl2·2H2O
      0.6 g/L citrate Fe-NH4
      2 g/L Na2CO3
    3. A5 solution
      2.86 g/L H3BO3
      1.81 g/L MnCl2·4H2O
      0.31 g/L Na2-MoO4·2H2O
      0.22 g/L ZnSO4·7H2O
      0.05 g/L Co(NO3)·6H2O
      0.08 g/L CuSO4·5H2O
  2. Extraction buffer
    20 mM Tris-HCl, pH 8.0
    1 or 10 mM Mg-acetate
    20 mM NH4Cl
    5 mM β-mercaptoethanol (added just prior to use)
    This solution (lacking the β-mercaptoethanol) was autoclaved at 121 °C, 1 atm for 20 min
  3. Sucrose buffer 1
    20 mM Tris-HCl, pH 8.0
    1 or 10 mM Mg-acetate
    20 mM NH4Cl
    10% (w/v) sucrose
    5 mM β-mercaptoethanol (added just before use)
  4. Sucrose buffer 2
    20 mM Tris-HCl, pH 8.0
    1 or 10 mM Mg-acetate
    20 mM NH4Cl
    30% (w/v) sucrose
    5 mM β-mercaptoethanol (added just before use)
    Sucrose buffer 1 and 2 are prepared from autoclaved RNase free stocks and sucrose was dissolved at the end
  5. 60% (w/v) sucrose
    Prepared with Milli-Q water and heating. Once the sucrose is dissolved the volume is adjusted with a test tube
  6. 100% (w/v) TCA solution
    To a previously unopened bottle containing 500 g of TCA, add 227 ml of sterile Milli-Q water. The resulting solution will contain 100% (w/v) TCA
  7. 80% (v/v) glycerol
    Prepared with Milli-Q water and autoclaved at 121 °C, 1 atm for 20 min

Note: All solutions are stored at 4 °C.

Acknowledgments

This protocol was adapted and modified from previously published studies by Sugita et al. (2000). We are grateful to Dr. J. de la Cruz for his expert advice, helpful discussions and a critical reading of the manuscript. We thank M. Roldán for technical assistance. This work was supported by Junta de Andalucía (grant P07-CVI-02792 and group BIO-284) and Spanish Ministerio de Economía y Competitividad (MINECO) and Fondo Social Europeo (FSE) (grant BFU2013-41712-P).

References

  1. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  2. Galmozzi, C. V., Florencio, F. J. and Muro-Pastor, M. I. (2016). The cyanobacterial ribosomal-associated protein LrtA is involved in post-stress survival in Synechocystis sp. PCC 6803. PLoS One 11(7): e0159346.
  3. McKinney, G. (1941). Absorption of light by chlorophyll solution. J Biol Chem 140:315-21.
  4. Mutsuda, M. and Sugiura, M. (2006). Translation initiation of cyanobacterial rbcS mRNAs requires the 38-kDa ribosomal protein S1 but not the Shine-Dalgarno sequence: development of a cyanobacterial in vitro translation system. J Biol Chem 281(50): 38314-38321.
  5. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. and Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111: 1-61.
  6. Sato, N., Wada, A. and Tanaka, A. (1998). Ribosomal proteins in the cyanobacterium Anabaena variabilis strain M3: presence of L25 protein. Plant Cell Physiol 39(12): 1367-1371.
  7. Sugita, C., Sugiura, M. and Sugita, M. (2000). A novel nucleic acid-binding protein in the cyanobacterium Synechococcus sp. PCC6301: a soluble 33-kDa polypeptide with high sequence similarity to ribosomal protein S1. Mol Gen Genet 263(4): 655-663.

简介

核糖体颗粒的分离是研究核糖体组分以及与核糖体相互作用以调节蛋白质合成并调节细胞表达谱的反式因子的分析中必不可少的步骤响应不同的环境条件。在本协议中,我们描述了从单细胞蓝细菌集胞藻分离70S核糖体的过程。 PCC 6803(以下简称集胞藻)。我们已经成功地使用这个方案来研究蓝细菌核糖体相关蛋白LrtA,它是细菌HPF(冬眠促进因子)的同系物(Galmozzi等人,2016)。

背景 据报道蓝藻核糖体几乎没有生物化学研究。已经通过差速离心分离70S核糖体颗粒,然后通过二维电泳分析核糖体蛋白质(Sato et al。,et al。 ,1998)。核糖体也已经从聚球藻(Spechococcus)进行制备。 PCC 6301细胞使用组合差速离心和蔗糖步骤梯度的方案(Sugita等人,2000)。通过差速离心分离细胞提取物也被用于制备核糖体样品用于在不同的聚球藻菌株中开发体外翻译系统(Mutsuda和Sugiura,2006 )。基于针对聚球藻(Sugita等人,2000)所述的方法,本文所述的针对集胞藻的方法允许使用以下方式纯化核糖体颗粒线性蔗糖梯度的超速离心。

关键字:核糖体, 蔗糖梯度, 集胞藻, 蓝细菌, 光合原核生物

材料和试剂

  1. 离心管,聚丙烯15 ml(Beckman Coulter,目录号:342082)
  2. 1.5ml微量离心管(Microtubes)(Corning,Axygen,目录号:MCT-150-C)
  3. 1.5ml聚苯乙烯分光光度计比色杯(KARTELL SPA CIA DELLE INDUSTRIE,目录号:1938)
  4. 超离心机薄壁聚集管13.2 ml(Beckman Coulter,目录号:331372)
  5. 0.45微米Millipore过滤器(MILLEX-HV PVDF)(EMD Millipore,目录号:SLHV033RS)
  6. A-5型氧化铝(Sigma-Aldrich,目录号:A2039)
    注意:本产品已停产。
  7. 微量吸头(200μl)(Daslab,目录号:162001X)
  8. 微量吸头(1000μl)(Daslab,目录号:162222X)
  9. 硅胶管(VWR,目录号:228-0713)
  10. 集胞藻 sp。 PCC 6803细胞(蓝藻的巴斯德培养物收集)
  11. Milli-Q水
  12. 蒸馏水
  13. 硝酸钠(NaNO 3)(AppliChem,目录号:141702)
  14. 碳酸氢钠(NaHCO 3)(AppliChem,目录号:141638)
  15. 磷酸氢二钾(K 2 HPO 4)(AppliChem,目录号:141512)
  16. 七水硫酸镁(MgSO 4·7H 2 O)(Duchefa Biochemie,目录号:M0513)
  17. 柠檬酸一水合物(C 6 H 8 O 7 H 2 O)(EMD Millipore,目录号: 100244)
  18. EDTA-Na 2(Duchefa Biochemie,目录号:E0511)
  19. 氯化钙二水合物(CaCl 2·2H 2 O)(Merck,目录号:2382)
  20. 柠檬酸盐Fe-NH 4(Sigma-Aldrich,目录号:F5879)
  21. 磷酸钠(Na 2 CO 3)(Merck,目录号:6392)
  22. 硼酸(H 3 3 BO 3)(EMD Millipore,目录号:100165)
  23. 四氢化锰(II)四水合物(MnCl 2·4H 2 O)(EMD Millipore,目录号:1059270)
  24. 钼酸钠脱水(Na 2 MoO 4·2H 2 O)(Merck,目录号:6521)
  25. 硫酸锌七水合物(ZnSO 4·7H 2 O)(Merck,目录号:8883)
  26. 硝酸钴六水合物(Co(NO 3 3)2·6H 2 O)(AppliChem,目录号:131258)
  27. 硫酸铜(II)五水合物(CuSO 4·5H 2 O)(EMD Millipore,目录号:102790)
  28. Tris Ultrapure(Duchefa Biochemie,目录号:T1501)
  29. 氯化铵(NH 4 Cl)(EMD Millipore,目录号:101145)
  30. 醋酸镁(Sigma-Aldrich,目录号:M5661)
  31. β-巯基乙醇(EMD Millipore,目录号:805740)
  32. 甘油(AppliChem,目录号:131339)
  33. 液氮
  34. 三氯乙酸(TCA)(EMD Millipore,目录号:100807)(仅限西方)
  35. 丙酮(EMD Millipore,目录号:100014)(仅限西方)
  36. 氯化铵(EMD Millipore,目录号:101145)
  37. 蔗糖(Sigma-Aldrich,目录号:S0389)
  38. 盐酸发烟47%(EMD Millipore,目录号:100317)
  39. 甲醇外推(Scharlau,目录号:ME0301005P)
  40. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A4503)
  41. 蛋白质测定染料试剂浓缩物(Bio-Rad Laboratories,目录号:5000006)
  42. BG11生长培养基(参见食谱)
  43. 提取缓冲液(见配方)
  44. 蔗糖缓冲液1(参见食谱)
  45. 蔗糖缓冲液2(参见食谱)
  46. 60%(w/v)蔗糖(见食谱)
  47. 100%(w/v)TCA溶液(参见食谱)
  48. 80%(v/v)甘油(参见食谱)

设备

  1. 荧光灯
  2. 锥形瓶250毫升(杜兰集团,目录号:21 771 36)
  3. 用于制备溶液的烧瓶和磁力搅拌器
  4. 轨道摇床(IKA,型号:KS501)
  5. Roux培养瓶1升(Sigma-Aldrich,目录号:CLS12901L)
  6. 玻璃瓶5升(Iso瓶蓝色瓶盖5000毫升)(Kavalierglass,Simax,目录号:CFB020)
  7. 天平(Sartorius,目录号:TE1502S和Precisa,型号:XT120A)
  8. pH计(HACH LANGE SPAIN,CRISON,型号:BASIC 20)
  9. 流量计(ABB,型号:A6200)
  10. 压缩空气泵
  11. 离心机(Beckman Coulter,型号:Avanti J-25)
  12. 刚性聚丙烯管(Fisher Scientific,目录号:11723904)
  13. 固定角转子(Beckman Coulter,型号:JLA-16.250,目录号:363934)
  14. 离心瓶聚丙烯250 ml(Beckman Coulter,目录号:356011)
  15. 瓷砂浆和杵(10厘米Ø)(费雪科学,目录号:S337621)
  16. 灭菌炉(JP SELECTA,目录号:2000381)
  17. 高压釜(三洋Labo高压灭菌器)(松下生物医学,型号:MLS-3020U)
  18. 自动微量移液管
  19. 涡流搅拌机(Heidolph Instruments,型号:Heidolph Reax 2000)
  20. UV-VIS分光光度计(Thermo Electron,型号:BioMate 5)
  21. 固定角转子(Beckman Coulter,型号:JA-25.15)
  22. 梯度制造商(Hoefer,目录号:SG15)
  23. 蠕动泵(Pharmacia Biotech,型号:Pump P-1)
  24. 磁板搅拌器(Cole-Parmer,目录号:EW-04807-45)
  25. 超速离心机(Beckman Coulter,型号:XL-80)
  26. 摇摆转子(Beckman Coulter,型号:SW 41 Ti)
  27. 使用TRIS TM(Teledyne Isco)的ISCO UA-6检测器UV/VIS(密度梯度分馏器,Teledyne Isco,目录号:67-9000-176)
  28. 微量离心机(Eppendorf,型号:5424/5415 R)
  29. Thermoblock(Eppendorf Thermomixer compact)(仅限西方)
  30. 金属连接器(Parker-Legris,目录号:0180 04 00)
  31. CO <2>气瓶容器

程序

整个协议的概述分为四个步骤,如图1所示

图1.协议中描述的完整过程的方案

  1. 培养集胞藻
    1. 在补充有1g NaHCO 3 NaHCO 3的BG11培养基(Rippka等人,1979)中,在30℃下自发地生长集胞藻细胞, (BG11C)在连续照明下(50μmol光子m sec -1 ,来自荧光灯的白光)。
      1. 通常培养开始于用覆盖有棉花的锥形瓶中生长的预接种物(50ml),并放置在位于照明室中的轨道振荡器(100rpm)上。
      2. 然后将这些细胞用于在Roux烧瓶中接种750ml BG11C培养基。最后将文化缩放至5升瓶(不含盖子并覆盖棉花)。
        注意:为了达到最佳生长,将Roux烧瓶和5升瓶中的培养物用含CO 2的富含空气(1%v/v)鼓泡,使用刚性聚丙烯管,硅胶管道和金属连接器。压缩空气流驱动鼓泡,并使用流量计建立空气CO 2混合物。
    2. 生长阶段的估计
      使用体积为3-5L的细胞,根据每个实验的目的指数或静止生长期。通过测量细胞甲醇提取物中的总叶绿素含量来估计生长期(McKinney,1941)。
      1. 离心1毫升培养物在1.5毫升微量离心管中,12,000 x g。
      2. 弃去950μl上清液,并将沉淀重新悬浮在剩余的50μl中。加入950μl甲醇,剧烈旋转1 min。
      3. 以12,000 x g离心2分钟。通过测量上清液中665nm处的吸光度计算叶绿素浓度(E = 74.46ng -1 cm -1)。
      4. 具有2-4μg/ml总叶绿素的培养物被认为是指数期,超过5μg/ml的培养物被认为是静止的。
    3. 收获细胞
      1. 在JLA-16.250 Beckman转子中以9,605 x g离心在4℃离心10分钟。
      2. 将细胞合并在一个瓶子中,并估计鲜重。 2-5g的细胞已足够用于手术。

  2. 集胞藻细胞的破裂和分级
    1. 预处理
      将所有玻璃材料,瓷砂浆和杵在200℃的灭菌烘箱中加热3小时,所有塑料材料在121℃下高压灭菌20分钟。
      注意:无RNase的水用于制备所有缓冲液。
    2. 细胞破裂细胞 放在预先冷却的瓷砂浆中,同样重量的细胞和氧化铝。将冰沙放在冰桶的顶部。与杵混合,一旦细胞和氧化铝充分混合,继续研磨2分钟。在使用前立即加入约20ml的提取缓冲液(1或10 mM醋酸镁),加入β-巯基乙醇
    3. 细胞分裂细胞的分化 在两个15毫升的聚丙烯管中尽可能多地回收材料。在4℃下在JA-25.15转子中以10,000×g离心10分钟以除去氧化铝,细胞碎片和不间断的细胞。将上清液转移到新的15ml管中,并在4℃下在同一转子中以30,000×g离心30分钟。这种离心是完全消除膜所必需的。
    4. 冻结储备
      在具有5%(v/v)甘油的1.5ml Eppendorf管中,从该离心步骤(S30级分)的上清液中分配等分试样。对于该过程,将1.5ml管置于冰上,并向每个管中加入40μl80%(v/v)甘油和600μlS30级分。使用微量移液器轻轻混合,并迅速冷冻管中液氮。在冷冻之前,保留一份等分试样以Bradford的标准方法测定该S30级分的总蛋白量,使用白蛋白牛作为参考(Bradford等人,1976)。通常在S30级分中获得总蛋白浓度为10-15μg/μl。
      注意:
      1. 当提取缓冲液和梯度溶液含有1mM Mg-醋酸盐(低Mg 2+)时,大多数细菌核糖体被分解成30S和50S亚基,但当使用10mM Mg-醋酸盐(高Mg 2+)时,在缓冲液和溶液中,70S空位核糖体峰主要观察。
      2. 值得注意的是,在蓝细菌中,藻胆蛋白占蛋白质总浓度的约30%。在考虑本程序的下一步所需的S30分数的数量时,必须考虑到这一点。
      3. S30分数可以在-80℃下以不同的等分试样储存,以在不同的日子加载分离的梯度。这是备份,以防在协议的后续步骤中存在任何问题,特别是在离心,分馏或生成梯度轮廓时。

  3. 蔗糖梯度的制备和超速离心
    1. 蔗糖梯度的制备
      1. 使用连接到以120ml/h工作的蠕动泵(P1)的线性梯度制备器制备10-30%蔗糖梯度。使用蔗糖缓冲液1和2溶液(1或10 mM醋酸镁),使用前加入β-巯基乙醇和13.2 ml超离心机薄壁聚集管。将梯度仪置于磁力搅拌板的顶部。
      2. 在与泵连接的梯度制造器室中引入小型磁力搅拌棒。在该隔室中倒入5.6ml蔗糖缓冲液1,并在梯度制备器的另一个室中倒入5.6ml蔗糖缓冲液2。蠕动泵的出口连接到导入梯度管的玻璃毛细管。
      3. 连接梯度制造器的两个隔间,同时打开泵P1。
      4. 当气泡进入梯度时,当梯度制造商中引入的溶液耗尽时,停止泵。
      5. 然后缓慢从梯度管中取出毛细管,以免干扰。
      6. 将管在4℃下储存约8小时,然后使用以允许线性蔗糖梯度的平衡。
      7. 加载前,通过一个0.45μm的过滤器过滤S30分数。
    2. 超速离心
      1. 使用微量移液管,并通过接近蔗糖梯度表面的单滴,轻轻地将S30级分(100-300μl)层压在10-30%蔗糖梯度的顶部。
      2. 在SW 41 Ti转子的预冷Beckman超速离心机中,在77,175 x g离心机在2℃下放置8小时。

  4. 核糖体颗粒的分离
    1. 仔细地从离心机中取出梯度管,并在将其放入ISCO分馏系统之前用Milli-Q水完全填充每个管。
    2. 使用60%(w/v)蔗糖从顶部分离梯度以将管的内容物推向UV检测器(A 254),使用TRIS TM (流速1ml/min)。打开记录仪(1厘米/分钟,灵敏度1)以获得蔗糖梯度曲线,同时手动收集级分(0.5毫升)。
    3. 将收集的液体置于含有液氮的容器中,以迅速冷冻,然后在-80°C下储存,直至使用。
      预期配置文件的代表性示例如图2所示

      图2.显示游离亚基和70S空位核糖体的沉积曲线。将30个上清液在10-30%蔗糖密度梯度上在1mM( A)或10mM(B)Mg 2+。小数字是指收集的分数。改编自(Galmozzi等人,2016年)。

    4. 从这一点上对这些部分的处理取决于将给予它们的用途。在通过蛋白质印迹分析的情况下,用100%(w/v)TCA沉淀总蛋白(参见食谱)。
      1. 向每个级分(10%TCA最终浓度)中加入55μl该溶液,在冰上孵育30分钟,并在最大速度和4℃下在微量离心机(Eppendorf 5415R)中离心10分钟。
      2. 弃去上清液,加入1毫升-20℃冷冻的丙酮至每个级分颗粒中,以除去残余的TCA。
      3. 仔细取出丙酮,不影响颗粒的完整性。
      4. 通过蒸发丙酮来干燥颗粒,将其在60℃下孵育成热块。
      5. 将沉淀重悬于无RNase的Milli-Q水中,并加入标准电泳方案的加载缓冲液。

笔记

目前来自ISCO UA-6检测器的模拟信号可以使用National Instruments的Bus-Powered Multifunction DAQ USB Device(NI USB-6008)转换成数字信号。

食谱

  1. BG11生长培养基(Rippka等人,1979)
    1. BG11C(1L)
      1.5g NaNO 3
      1g NaHCO 3
      0.2ml 1M K 2 HPO 4
      10 ml浓缩液BG11(见下文)
      高压灭菌在121°C,1 atm 20分钟
    2. 集中BG11
      7.5g/L MgSO 4·7H 2 O→// 0.6 g/L柠檬酸
      0.1g/L EDTA-Na 2
      100 ml A5溶液(见下文)
      3.6g/L CaCl 2·2H 2 O
      0.6g/L柠檬酸盐Fe-NH 4
      2g/L Na 2 CO 3
    3. A5解决方案
      2.86g/L H 3 3< 3< 3>
      1.81g/L MnCl 2·4H 2 O -/- 0.31g/L Na 2 -MoO 4·2H 2 O
      0.22g/L ZnSO 4·7H 2 O
      0.05g/L Co(NO 3 3)·6H 2 O
      0.08g/L CuSO 4·5H 2 O
  2. 提取缓冲区
    20mM Tris-HCl,pH8.0
    1或10 mM醋酸镁 20mM NH 4 Cl
    5 mMβ-巯基乙醇(加入使用前)
    将该溶液(缺少β-巯基乙醇)在121℃,1大气压下高压灭菌20分钟
  3. 蔗糖缓冲液1
    20mM Tris-HCl,pH8.0
    1或10mM醋酸镁 20mM NH 4 Cl
    10%(w/v)sacarose
    5 mMβ-巯基乙醇(加入使用前)
  4. 蔗糖缓冲液2
    20mM Tris-HCl,pH8.0
    1或10 mM醋酸镁 20mM NH 4 Cl
    30%(w/v)sacarose
    5 mMβ-巯基乙醇(加入使用前)
    蔗糖缓冲液1和2由高压灭菌的无RNA酶的原料制备,蔗糖在末端溶解
  5. 60%(w/v)蔗糖
    用Milli-Q水和加热液制备。蔗糖溶解后,用试管调节体积
  6. 100%(w/v)TCA溶液
    向含有500g TCA的先前未开封的瓶中加入227ml无菌Milli-Q水。所得到的溶液将含有100%(w/v)TCA
  7. 80%(v/v)甘油
    用Milli-Q水准备,并在121℃,1大气压下高压灭菌20分钟

注意:所有解决方案都保存在4°C。

致谢

该协议是由Sugita等人先前发表的研究进行了修改和修改的。 (2000)。我们非常感谢J. de la Cruz博士的专家意见,有用的讨论和对稿件的批判性阅读。我们感谢M.Roldán的技术援助。这项工作得到了安达卢西亚军政府(授予P07-CVI-02792和BIO-284组)和西班牙经济部长(MINECO)和Fondo Social Europeo(FSE)(授权BFU2013-41712-P)的支持。

参考文献

  1. Bradford,MM(1976)。  一种快速敏感的方法用于利用蛋白质 - 染料结合原理定量微量的蛋白质。
    Anal Biochem 72:248-254。
  2. Galmozzi,CV,Florencio,FJ和Muro-Pastor,MI(2016)。  蓝细菌核糖体相关蛋白LrtA参与集胞藻的后应激存活。 PCC 6803. PLoS One 11(7):e0159346。
  3. McKinney,G.(1941)。  吸收光叶绿素溶液。生物化学 140:315-21。
  4. Mutsuda,M。和Sugiura,M。(2006)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/17046824"target ="_ blank" >蓝芽细胞rbcS mRNA的翻译启动需要38kDa的核糖体蛋白S1,而不是Shine-Dalgarno序列:在体外翻译系统中开发蓝细菌。/em> 281(50):38314-38321。
  5. Rippka,R.,Deruelles,J.,Waterbury,JB,Herdman,M.and Stanier,RY(1979)。< a class ="ke-insertfile"href ="http://mic.microbiologyresearch.org/内容/journal/micro/10.1099/00221287-111-1-1"target ="_ blank">蓝细菌纯培养物的通用分配,菌株历史和性质。 Gen Microbiol 111 :1-61。
  6. Sato,N.,Wada,A.and Tanaka,A。(1998)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/10050320"目标="_ blank">蓝色杆菌中的核糖体蛋白质欧洲斑马鱼M3株:存在L25蛋白。植物细胞生理39(12):1367-1371 。
  7. Sugita,C.,Sugiura,M.and Sugita,M。(2000)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/10852488" target ="_ blank">蓝细菌聚球蓝细菌中的新型核酸结合蛋白。 PCC6301:与核糖体蛋白S1具有高度序列相似性的可溶性33-kDa多肽。 Mol Gen Genet 263(4):655-663。
  • 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: © 2017 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. Galmozzi, C. V. and Muro-Pastor, M. (2017). Isolation of Ribosomal Particles from the Unicellular Cyanobacterium Synechocystis sp. PCC 6803. Bio-protocol 7(6): e2176. DOI: 10.21769/BioProtoc.2176.
  2. Galmozzi, C. V., Florencio, F. J. and Muro-Pastor, M. I. (2016). The cyanobacterial ribosomal-associated protein LrtA is involved in post-stress survival in Synechocystis sp. PCC 6803. PLoS One 11(7): e0159346.
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