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Peer-reviewed

Affinity Purification of the RNA Degradation Complex, the Exosome, from HEK-293 Cells

来自HEK-293细胞的外泌体RNA降解复合物的亲和纯化

MD Michal Domanski
John LaCava John LaCava

发布: 2017年04月20日第7卷第8期 DOI: 10.21769/BioProtoc.2238 浏览次数: 8525

评审: Gal HaimovichAnonymous reviewer(s)

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Abstract

The RNA exosome complex plays a central role in RNA processing and regulated turnover. Present both in cytoplasm and nucleus, the exosome functions through associations with ribonucleases and various adapter proteins (reviewed in [Kilchert et al., 2016]). The following protocol describes an approach to purify RNA exosome complexes from HEK-293 cells, making use of inducible ectopic expression, affinity capture, and rate-zonal centrifugation. The obtained RNA exosomes have been used successfully for proteomic, structural, and enzymatic studies (Domanski et al., 2016).

Keywords: RNA exosome (RNA外泌体) search EXOSC10 (EXOSC10) search Cryomilling (低温球磨) search HEK-293 suspension culture (HEK-293悬浮培养) search Affinity capture (亲和捕获) search Rate-zonal centrifugation (速率区带离心) search

Background

In our previous work, we established an isogenic HEK-293 cell line expressing C-terminally 3xFLAG-tagged exosome component EXOSC10 (RRP6) under the control of a tetracycline-inducible CMV promotor (HEK-293 Flp-In T-REx – Thermo Fisher Scientific). This system permitted us to express the tagged EXOSC10 protein at a level comparable to the endogenous WT protein, and to explore exosome purification protocols using a magnetic anti-FLAG affinity medium and protein extracts derived from cryomilled cell powder (Domanski et al., 2012). Further exploring the protein extraction conditions used, we developed a protocol permitting the retention of DIS3 (RRP44) within affinity captured exosomes, which has otherwise proven difficult (Hakhverdyan et al., 2015). Building on these studies, we further purified RNA exosomes, +/- DIS3, by rate-zonal centrifugation using glycerol density gradients (Domanski et al., 2016). Although the presence of the detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) in the protein extract enhanced the yield of DIS3 co-purifying with affinity captured exosomes, the interaction was subsequently lost during sedimentation in a glycerol density gradient. To counteract this, the crosslinker DTSSP [3,3’-dithiobis(sulfosuccinimidyl propionate)] was employed. The treatment enabled the retention of DIS3 within exosomes during sedimentation, but negatively affected DIS3 enzymatic functions. The peak fractions from DIS3 +/- exosome fractions both contained apparent exoribonucleolytic activities consistent with EXOSC10-derived distributive 3’-5’ hydrolysis. Apparent structural differences between samples that retained DIS3 (DTSSP-treated) and those that did not could be observed by negative stain electron microscopy. The protocol presented here will enable users to obtain endogenously assembled RNA exosome fractions suitable for additional analytical methods including in vitro biochemistry, enzymology, and electron microscopy. Note that many aspects of this protocol can be easily adapted, e.g., to use (1) different affinity tags and expression contexts, or (2) antibodies against the endogenous protein (LaCava et al., 2015).

Materials and Reagents

Note: Catalog numbers are given for most of the reagents listed below; an equivalent quality reagent from an alternative supplier can typically be substituted with comparable results. Standard materials and reagents for mammalian cell culture are required and are not all explicitly listed below.

  1. Pipette tips 
  2. Nunclon cell culture 245 mm (500 cm2) square dish (Sigma-Aldrich, catalog number: D8679 )
  3. Nunclon 175 cm2 cell culture flask (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 156502 )
  4. 15” cell scraper (Fisher Scientific, catalog number: 08-100-242 )
  5. 50 ml polypropylene conical tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339652 )
  6. 20 ml Luer-lock syringe (BD, catalog number: 302830 )
  7. Parafilm (Sigma-Aldrich, catalog number: P7793 )
  8. Syringe end caps (Bio-Rad Laboratories, catalog number: 7311660EDU )
  9. 2 ml microcentrifuge tubes (e.g., Eppendorf, catalog number: 022363344 )
  10. 0.45 μm polyethersulfone (PES) sterile syringe filters (VWR, catalog number: 28145-505 )
  11. 5 ml Ultracentrifuge tubes (Seton Scientific, catalog number: 7022 )
    Note: If you will use the BioComp Instruments Piston Gradient Fractionator to recover fractions of purified exosomes (see Equipment), we recommend you obtain these tubes from BioComp because they are tolerance tested for compatibility.
  12. HEK-293 Flp-In T-REx EXOSC10-3xFLAG cells ([Domanski et al., 2016]; available upon request)
  13. DMEM, high glucose, GlutaMAX (Thermo Fisher Scientific, catalog number: 31966047 )
  14. Fetal bovine serum (FBS), tetracycline-free
    Note: Numerous suppliers can provide this. Many suppliers carry FBS products not labelled as tetracycline-free, but consulting the product specification sheet for a given lot may reveal that tetracycline has been tested for and found to be absent. In our hands, the performance of such lots has been identical to ‘certified’ tetracycline-free FBS.
  15. 100x penicillin-streptomycin (P/S) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  16. Trypsin-EDTA, 0.05% (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
  17. Tetracycline (Sigma-Aldrich, catalog number: 87128 )
    Note: Prepare a stock solution of 10 mg/ml in ethanol and store at -20 °C. The working solution is 5 μg/ml–for the induction add 1 μl per 1 ml cell culture medium. Doxycycline can also be used.
  18. Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher Scientific, catalog number: 10010023 )
  19. 100x L-glutamine (200 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  20. TrypLE dissociation reagent (Thermo Fisher Scientific, GibcoTM, catalog number: 12605010 )
  21. Phenol red solution, 0.5% (Sigma-Aldrich, catalog number: P0290 )
  22. Liquid nitrogen (LN2)
  23. Freestyle 293 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 12338026 )
  24. 3 M ammonium sulfate in 0.1 M sodium phosphate buffer pH 7.4
  25. Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S )
  26. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  27. 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: 54457 )
  28. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  29. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 71687 )
  30. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 320331 )
  31. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  32. Protease inhibitor cocktail, EDTA-free (Roche Diagnostics, catalog number: 11873580001 )
  33. CHAPS (Sigma-Aldrich, catalog number: C5070 )
  34. Anti-FLAG M2 antibody (Sigma-Aldrich, catalog number: F3165 or F1804 )
  35. Dynabeads M-270 epoxy (Thermo Fisher Scientific, InvitrogenTM, catalog number: 14302D )
  36. 0.1 M sodium phosphate buffer pH 7.4
  37. Tris base (Sigma-Aldrich, catalog number: 93362 )
  38. 3xFLAG peptide (Sigma-Aldrich, catalog number: F4799 ) reconstituted at 5 mg/ml in TBS (50 mM Tris-HCl pH 7.4, 150 mM NaCl). Aliquots should be stored at -20 °C
  39. DTSSP (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 21578 )
  40. 4x LDS (lithium dodecyl sulfate) sample loading buffer (Thermo Fisher Scientific, NovexTM, catalog number: NP0007 )
  41. 10x sample reducing agent (Thermo Fisher Scientific, NovexTM, catalog number: NP0004 ); or 500 mM dithiothreitol (DTT)
  42. 4-12% Bis-Tris 26-well midi gels (Thermo Fisher Scientific, InvitrogenTM, catalog number: WG1403BOX )
  43. 20x MOPS (3-morpholinopropane-1-sulfonic acid) gel running buffer (Thermo Fisher Scientific, NovexTM, catalog number: NP0001 )
  44. SilverQuest Silver Staining Kit (Thermo Fisher Scientific, NovexTM, catalog number: LC6070 )
  45. Sypro ruby protein gel stain (Sigma-Aldrich, catalog number: S4942 )
  46. Anti-EXOSC10 antibody (Abcam, catalog number: ab95028 )
  47. Anti-SKIV2L2 antibody (Abcam, catalog number: ab70552 )
  48. Anti-EXOSC3 antibody (Proteintech, catalog number: 15062-1-AP )
  49. ExoI extraction solution (see Recipes)
  50. ExoII extraction solution (see Recipes)
  51. Gradient solution – ‘light’ (see Recipes)
  52. Gradient solution – ‘heavy’ (see Recipes)

Equipment

Note: Catalog numbers are given for most of the equipment listed below; instruments from alternative manufacturers may be substituted provided equivalent functionality.

  1. Pipettes 
  2. Balance
  3. CO2 incubator for mammalian cell culture
  4. Refrigerated microcentrifuge (capable of reaching 20,000 x g)
  5. New Brunswick Innova 2000 platform shaker (Eppendorf, New BrunswickTM, model: Innova® 2000 , catalog number: M1190-0002)
    Note: Any shaker installed in a mammalian cell culture incubator must be able to tolerate continuous high humidity (~90% relative humidity).
  6. 250 ml plastic beaker
  7. Hemocytometer
  8. 1 L square PYREX bottles (Corning, PYREX®, catalog number: 1396-1L )
  9. Milling balls, stainless steel, 20 mm (Retsch, catalog number: 05.368.0062 )
  10. 50 ml stainless steel milling jar (Retsch, catalog number: 01.462.0149 )
  11. Metal spatula
  12. Planetary Ball Mill PM 100 (Retsch, model: PM 100 , catalog number: 20.540.0001)
  13. Thermomixer (Eppendorf, model: Thermomixer® R , catalog number: 5355000.011; or equivalent)
  14. Vortex mixer equipped with head for multiple 1.5/2.0 ml tubes (Thermo Fisher Scientific, Fisher Scientific, model: Fisher ScientificTM Vortex Mixer , catalog number: 02-215-386)
  15. Neodymium magnet microfuge tube rack (Thermo Fisher Scientific, catalog number: 12321D )
  16. Ultracentrifuge compatible with either of the rotors listed below (Beckman Coulter), e.g., Optima L or Optima MAX series (Beckman Coulter, model: Optima L or Optima MAX series )
  17. SW 55 Ti or MLS-50 rotor (Beckman Coulter, model: SW 55 Ti or MLS-50 )
  18. Microtip sonicator (e.g., Qsonica, model: Q700 ) equipped with a low intensity 1/16” microtip probe (Qsonica, catalog number: 4717)
  19. Gradient fractionator and accessories for SW 55 Ti (BioComp Instruments, catalog number: 152-001 )
  20. Gradient master and accessories for SW 55 Ti (BioComp Instruments, catalog number: 107-201M )

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

Domanski, M. and LaCava, J. (2017). Affinity Purification of the RNA Degradation Complex, the Exosome, from HEK-293 Cells. Bio-protocol 7(8): e2238. DOI: 10.21769/BioProtoc.2238.

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分类

生物化学 > 蛋白质 > 分离和纯化

分子生物学 > RNA > RNA 降解

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