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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

CG Carla V. Galmozzi
M M. Isabel Muro-Pastor

发布: 2017年03月20日第7卷第6期 DOI: 10.21769/BioProtoc.2176 浏览次数: 8432

评审: Dennis NürnbergAksiniya AsenovaManuel Miras Marin

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Jul 2016

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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) search Sucrose gradients (蔗糖梯度) search Synechocystis (集胞藻) search Cyanobacteria (蓝细菌) search Photosynthetic prokaryotes (光合原核生物) search

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

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文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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. I. (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.

    3. ris ris Download Citation in RIS Format

分类

微生物学 > 微生物生物化学 > 蛋白质

微生物学 > 微生物生物化学 > RNA

生物化学 > 蛋白质 > 分离和纯化

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