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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   

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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), EXOSC10(EXOSC10), Cryomilling(低温球磨), HEK-293 suspension culture(HEK-293悬浮培养), Affinity capture(亲和捕获), Rate-zonal centrifugation(速率区带离心)

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: 4417 )
  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

Note: Many of the steps described below for harvesting cells, milling them to powder, and carrying out an affinity capture can be viewed in our online video protocol (LaCava et al., 2016).

  1. Cell culturing and harvesting
    The tetracycline inducible EXOSC10-3xFLAG HEK-293 cell line can be obtained by contacting the authors or by following the manufacturer’s instructions. Except where noted, standard mammalian cell culturing procedures apply (Freshney 2011; Uphoff and Drexler, 2013).
    1. Adherent cell growth
      1. Seed ~107 cells on each of 16 x 500 cm2 square dishes in 90 ml DMEM supplemented with 10% v/v FBS and 1x P/S (DMEM-FBS-P/S).
        Note: 16 x 500 cm2 square dishes should yield at least 10 g of cell pellet, wet cell weight (WCW).
      2. When the cells reach ~90% confluency, remove the medium and induce the expression of EXOSC10-3xFLAG by adding fresh DMEM-FBS-P/S supplemented with 5 ng/ml tetracycline (Tet).
      3. Harvest the cells at 24 h post induction by scraping in ice-cold PBS–we use 30 ml/plate.
        Note: Alternatively, the cells can be harvested by trypsinization. For each plate, wash the cells with 20 ml PBS, add 10 ml of 0.05% trypsin-EDTA, tilt the dish to expose the cell monolayer, remove the excess liquid, and place them back into the CO2 incubator for ~5 min. Resuspend the cells in 10 ml DMEM-FBS-P/S and transfer to a 50 ml tube.
      4. Centrifuge the harvested cells at 1,000 x g for 5 min (4 °C).
      5. Discard the supernatant, resuspend, and combine the pellets in ice-cold PBS (~100 ml total volume), within two 50 ml tubes.
      6. Centrifuge at 1,000 x g for 5 min (4 °C).
      7. Discard the supernatant, resuspend the pellets in ice-cold PBS (~1 volume of PBS per volume of the pellet), and transfer the solution to a 20 ml capped syringe. Seal upper opening of the syringe with Parafilm (to avoid any accidental spillage) and place it inside a 50 ml tube.
      8. Centrifuge at 1,000 x g for 5 min (4 °C).
      9. Remove any remaining liquid, replace the plunger, and carefully inject the content into LN2.
        Note: First, fill 250 ml plastic beaker (placed inside a Styrofoam box) with LN2, then inject the cells directly into LN2.
      10. Store at -80 °C prior to cryomilling.
    2. Suspension cell growth
      For this protocol you will need a platform shaker with appropriate tolerances, installed inside of a 37 °C, humidified, 8% CO2 incubator. Suspension cell growth offers a convenient and cost effective way to generate large quantities of cells and maintain continuous cultures. The protocol presented here is adapted from a method we previously adapted (Muller et al., 2005) and applied to HEK-293TLD cells (Dai et al., 2012; Taylor et al., 2013; Taylor et al., 2016). Using HEK-293 Flp-In T-REx cells, suspension growth may yield ~3-5 million cells per ml, with growth slowing above ~3 million/ml. Obtaining accurate cell counts can be challenging because these suspensions tend to grow in clumps and require trituration by pipetting to be counted in a hemocytometer. Yields in WCW should be ≥ 4 g per 400 ml culture of cells expressing 3xFLAG-tagged EXOSC10 at near the endogenous level.
      1. Seed cells from stocks in the standard way and obtain 2 x 175 cm2 flasks of nearly confluent (90%+) cells. We use 25 ml of medium per 175 cm2 flask.
      2. Split the cells into 4 x 175 cm2 flasks in a 1:1 mixture of DMEM-FBS-P/S and Freestyle medium supplemented with 2% v/v FBS, 2 mM L-glutamine, and 0.5x P/S (the latter, referred to as conditioning medium). This initiates conditioning of adherent cells for suspension growth.
        Note: We have been unable to inoculate new suspension cultures directly from frozen cell stocks of suspension conditioned HEK-293 Flp-In T-REx cells. Therefore, we freshly condition cells for each new suspension growth, and then maintain cells in suspension for the needed duration. The difficulty in inoculating directly to suspension and the lower maximal cell density observed for HEK-293 Flp-In T-REx cells compared to HEK-293TLD cells may be due to the absence of the SV40 large T antigen in the former (Ahuja et al., 2005; Taylor et al., 2016).
      3. At the next cell splitting use a 1:3 mixture of DMEM-FBS-P/S and conditioning medium, transfer the cells into 8 x 175 cm2 flasks and grow to near confluence.
      4. Wash the cells with 10 ml PBS and discard the wash, then release the cells from the flasks using 0.1x TrypLE (1:10 dilution in PBS). Add 1.5 ml of 0.1x TrypLE solution, tilt each flask to expose the cell monolayer, and then remove the excess liquid. Incubate the flasks at RT for 10 min.
      5. Resuspend the released cells from each flask in 10 ml of conditioning medium. Combine 40 ml of the cell suspension and 60 ml of conditioning medium supplemented with 200 μl of 0.5% w/v phenol red (final 0.001% w/v) in a 1 L square glass bottle. This typically results in two 100 ml suspension cultures at a cell density of around a couple million per ml. Suspension cultures should be inoculated at between ~0.5-3 million cells/ml. Leave the bottle caps one-full-turn open (from just-closed) and set the cultures shaking at 130 RPM within the incubator.
        Note: The phenol red (PR) functions as a pH indicator and an indirect method of monitoring cell density in culture, complementing cell counting. The solution should have a light red appearance similar to commercially available DMEM, which also frequently contains this additive. The working volume for 1 L glass bottles in this protocol is between 100-400 ml. Two 400 ml cultures should ultimately yield at least 8 g WCW.
      6. Monitor the culture by cell counting and medium color. When the cell density is very near or exceeds 3 million cells/ml and begins to lighten in color towards orange, dilute the culture to 1-2 million cells/ml with 100 ml of Freestyle medium supplemented with 2 mM L-glutamine, 0.5x P/S, and 0.001% PR (no FBS)–resulting in a 1% final FBS concentration while other reagent concentrations remain the same (referred to as suspension medium). All future dilutions can be made with suspension medium.
        Note: Cells are typically round, single, and relatively easy to count for a few days after the initial inoculation, and then will become increasingly clumpy, even adhering to some extent to the side of the flask where the liquid line reaches.
      7. Continue to monitor the culture by cell counting and medium color. When the cell density is very near or exceeds 3 million cells/ml and begins to lighten in color towards orange, dilute the cultures to ≤ 3 million cells/ml with 200 ml suspension medium.
      8. Once this 400 ml of medium is very near or exceeds 3 million cells/ml, remove 50 ml of the culture from each and use this to seed two or more new 100 ml cultures at ~0.5-2 million cells/ml (if desired). To the remaining 350 ml of culture, add 50 ml of suspension medium supplemented with 8 ng/ml Tet (1 ng/ml final), and incubate overnight (~16-24 h).
        Note: We observed that 1 ng/ml of tetracycline in suspension culture was sufficient to give expression comparable to 5 ng/ml in adherent growth. However, we encourage researchers to test expression in their hands by carrying out an anti-RRP6 Western blot after induction (Domanski et al., 2012). This protocol can be miniaturized using smaller culture vessels for pre-tests of that nature.
      9. Harvest the cells by centrifugation, wash and freeze in LN2 as described in steps d-j in section A1–Adherent cell growth–above.

  2. Cryomilling procedure
    The cryogenic disruption of the mammalian cell pellets has been previously described (Domanski et al., 2012; Taylor et al., 2016; LaCava et al., 2016). Briefly, pre-cool a milling jar, 2 x 20 mm grinding balls, a metal spatula, and a 50 ml tube by immersing in LN2. Remove any LN2 from the jar chamber and place the cell pellets inside. Set the counter balance and clamp the jar in the Retsch PM 100. Run the machine for 3 milling cycles of 3 min each (reverse rotation, 1 min interval, no break time) at 400 RPM. Cool down the milling jar with LN2 between cycles. Recover the resulting cell powder using a spatula and transfer to 50 ml tube. Store at -80 °C.

  3. Coupling of the anti-FLAG M2 antibodies to Dynabeads M-270 epoxy
    The production of anti-FLAG magnetic medium has been previously described in detail (Cristea and Chait, 2011; Domanski et al., 2012; Taylor et al., 2016) and can also be carried out according to the manufacturer’s instructions (Thermo Fisher Scientific) with comparable results. Briefly, prepare 20 μl of 0.5 μg/μl antibody, in a sodium phosphate (pH 7.4) buffered 1 M ammonium sulfate solution, per mg of magnetic beads to be antibody-conjugated (i.e., 10 μg of antibody, in 20 μl solution, will be used per mg of beads). Combine the antibody solution and the magnetic beads and incubate overnight (16-24 h) at 30-37 °C with rotation. The mixing applied should be sufficient to ensure the beads remain in suspension for the duration of the incubation. For small batches, conjugation can be performed at 37 °C in a thermomixer (≥ 1,200 RPM). After coupling, remove the excess antibody solution, wash the beads to remove residual uncoupled antibodies, and finally resuspend the antibody-conjugated magnetic medium in 1x PBS [final] containing 0.5 mg/ml BSA and 50% v/v glycerol (storage solution): add 6.7 μl of storage solution per 1 mg of medium (resulting in a slurry of ~15% w/v) and store at -20 °C. The slurry can be stored in this way for at least 1 year without any noticeable loss of performance.

  4. Affinity purification
    A general procedure describing and demonstrating best practices for affinity purification using mammalian cell powder and magnetic media has been previously described (LaCava et al., 2016). The following implementation has been optimized for obtaining human RNA exosomes. Here, DIS3- exosomes are referred to as ExoI and the DIS3+ exosomes are ExoII.
    1. Pre-cool a metal spatula and 4 x 2 ml safe-lock tubes in LN2.
    2. Weight out 4 x 250 mg of HEK-293 EXOSC10-3xFLAG cell powder.
      Note: It is important to keep the cell powder frozen. Leave the tubes in LN2 until the next step is initiated.
    3. Place the samples in the rack, open the caps, and let them stand at room temperature for 1 min.
    4. Add 1,250 μl ExoI or ExoII solution to each (see Recipes).
      Note: ExoI solution yields DIS3- exosomes, whereas ExoII yields DIS3+ exosomes.
    5. Vortex at maximum speed to fully resuspend the cell powder (should not require more than 30 sec), and immediately place the samples on ice.
      Note: Once the cell powder is resuspended, the sample should be held on ice between all manipulations throughout the procedure unless otherwise stated.
    6. Briefly sonicate to completely disperse and homogenize the cell powder. Use 4 pulses, 2 sec each (~30 J total per 250 mg sample) (LaCava et al., 2016).
      Note: If clumps are visible, the sonication step should be repeated until no clumps are obvious by visual inspection.
    7. Clarify the extract by centrifugation at 20,000 x g for 10 min (4 °C).
    8. While the samples are in the centrifuge, distribute 4x 25 μl anti-FLAG beads slurry into 2 ml microcentrifuge tubes and wash twice with 1 ml of the extraction solution of choice to thoroughly remove the storage solution.
      Note: We use 10 μl of the beads slurry per 100 mg of the cell powder. To wash the beads, combine 1 ml of extraction solution with 25 μl anti-FLAG magnetic medium (slurry) in a 2 ml microcentrifuge tube and vortex briefly to fully resuspend the beads. Pulse-spin the tube briefly in a mini-centrifuge to collect all the solution at the bottom and then place the tube in a magnetic tube rack until the beads are collected at the side of the tube. Remove the supernatant using a pipet or an aspirator, and repeat the washing step one more time. Perform both washing steps at room temperature. After washing, the beads may be held on ice and are ready for use. Alternatively, one can also wash a total of 100 μl anti-FLAG medium and distribute across 4 tubes after washing.
    9. Combine the clarified extracts with the pre-washed beads. Incubate for 1 h with rotation at 4 °C (cold room).
    10. Collect the beads on a magnet, remove the supernatant, and wash with 1 ml of ice cold extraction solution, in the manner noted above.
    11. Wash with extraction solution two more times. Move each sample to a fresh tube during the second wash.
      Note: Moving each sample (beads resuspended in extraction solution) to a fresh tube minimizes contamination of the eluate. Cell extract proteins non-specifically adsorbed to the internal surfaces of the tube used for affinity capture may be released into the sample during subsequent manipulations.
    12. After the 3rd wash, spin the samples briefly in a mini-centrifuge.
    13. Place again on a magnet and carefully remove any remaining liquid.
    14. Add 30 μl of elution solution (1 mg/ml 3xFLAG peptide diluted from the stock in extraction solution) to each tube.
    15. Incubate the samples at room temperature for 15 min with gentle agitation. ~60% power on a vortex equipped with a head for multiple 1.5/2.0 ml tubes.
      Note: Make sure the beads are not being mixed too vigorously, nor settling at the bottom of the tube.
    16. Place on a magnet and collect the supernatants.
    17. Add 30 μl of the extraction solution to wash the beads. Collect as above and pool the fractions together.
    18. Keep the samples on ice prior to loading on a gradient.

  5. DTSSP crosslinking to obtain ExoII
    DIS3 can be co-purified with the exosome in the ExoII extraction solution. However, it dissociates during sedimentation in a glycerol gradient. To preserve this interaction, we used chemical crosslinking. DTSSP will enhance the preservation of the DIS3/exosome interaction, but DIS3 enzymatic activity will be compromised by this treatment.
    1. Add 2 μl of freshly prepared 1.2 mM DTSSP per 10 μl of ExoII prepared by elution with 3xFLAG as described above.
    2. Carry out the crosslinking reaction for 50 min at RT.
    3. Quench by adding 2 μl 1 M Tris-HCl pH 8.
    4. Keep on ice prior to loading on a gradient.

  6. Rate zonal centrifugation
    The following settings and parameters are optimized for an SW 55 Ti rotor in a Beckman Optima L series ultracentrifuge. For MLS-50 details, refer to (Domanski et al., 2016).
    1. Prepare a 10-40% glycerol gradient using the BioComp Gradient Master instrument (short cap, 10-40% glycerol v/v) or other appropriate method. Always prepare an extra gradient to use as a balance.
    2. Pre-cool formed gradient to 4 °C (typically ≥ 1 h in a cold room).
    3. Load the sample(s) by gently pipetting onto the top of the gradient.
      Note: To avoid disrupting the top layer of the gradient, rest the pipette tip against the wall of the tube, just above the meniscus of the glycerol solution, and slowly eject the sample.
    4. Run the gradient using the following settings: minimum acceleration; no brake; 4 °C; 50k RPM; 6 h 36 min.
    5. After the run is completed, collect the fractions using a BioComp Piston Gradient Fractionator (consult the manufacturer’s instructions), or other appropriate method. We used the following settings with manual fraction collection: distance–2 mm, speed–0.3 mm/sec. This collection regime will produce a meniscus fraction, ~20 fractions of ~225 μl, and a bottom fraction. Some variations will be observed depending on the specific set-up implemented, however, using the described procedure and setup we retrieved the peak of sedimented ExoI in fraction 11 and ExoII in fractions 11 and 12.

  7. SDS-PAGE analysis of gradient fractions
    1. Mix 14 μl of the gradient fraction with 5 μl of 4x LDS and 1 μl of 10x reducing agent (or 500 mM DTT); heat at 70 °C for 10 min for ExoI (DIS3-) fractions or to 75 °C for 20 min for DTSSP-treated ExoII (DIS3+) fractions.
    2. Load the samples on a 26-well, 4-12% NuPAGE Bis-Tris gel, following the manufacturer’s instructions.
    3. Run at 200 V until the tracking dye reaches the bottom of the gel cassette.
    4. Remove the gel from the plastic cassette and place in a clean container.
    5. Stain with the silver or Sypro Ruby following manufacturers’ instructions.

Data analysis

During the sedimentation, free proteins (or partially assembled complexes) can be found close to the top of the gradient, whereas intact RNA exosomes migrate further and can be found around the middle of a gradient. Fractions containing intact exosomes exhibit signature staining intensities for (from the top of the gel) SKIV2L2, EXOSC10-3xFLAG, EXOSC9 and the core/low mass proteins, approximately distributed at the expected molecular masses (EXOSC9 runs closer to 60 kDa than its predicted mass of ~49 kDa). Figure 1A depicts a stained SDS-polyacrylamide gel loaded with gradient fractions obtained after ultracentrifugation (real data shown in Figure 1B). In this example, fraction number eleven (11) demonstrates the composition and staining intensities consistent with the peak fraction. The concentration of proteins present in gradient fractions should be sufficient for direct detection by e.g., silver or Sypro Ruby staining. Protein identities can be confirmed by Western blotting using specific antibodies (see Notes section) and/or mass spectrometry.


Figure 1. Representative results: RNA exosomes purified from HEK-293 cells expressing EXOSC10-3xFLAG. A. Schematic diagram of a stained SDS-polyacrylamide gel demonstrating protein bands consistent with the separation of EXOSC10-3xFLAG purified RNA exosomes obtained after sedimentation within a 10-40% v/v glycerol gradient. B. The original gel, reproduced from Domanski et al., 2016. Separated proteins were visualized by silver staining. The arrow indicates the peak fraction.

Notes

Listed are some commercially available antibodies that we have used with success to identify exosome components by Western blotting.

  1. Anti-EXOSC10 antibody (Abcam, catalog number: ab95028)
  2. Anti-SKIV2L2 antibody (Abcam, catalog number: ab70552)
  3. Anti-EXOSC3 antibody (Proteintech, catalog number: 15062-1-AP)

Recipes

  1. ExoI extraction solution
    20 mM HEPES-NaOH, pH 7.4
    300 mM NaCl
    1% Triton X-100 (v/v)
    1x protease inhibitor cocktail
  2. ExoII extraction solution
    20 mM HEPES-NaOH, pH 7.4
    100 mM NaCl
    5 mM CHAPS
    1x protease inhibitor cocktail
  3. 1 M Tris-HCl, pH 8.0 (for quenching DTSSP crosslinking reactions)
  4. Gradient solution – ‘light’
    10% v/v glycerol
    20 mM HEPES-NaOH, pH 7.4
    100 mM NaCl
    Sterile filter with a 0.45 μm syringe filter
  5. Gradient solution – ‘heavy’
    40% glycerol
    20 mM HEPES-NaOH, pH 7.4
    100 mM NaCl
    Sterile filter with a 0.45 μm syringe filter

Note: Many of the solutions used in the NuPAGE® system can be made in the laboratory and do not need to be purchased. Consult the recipes provided by the manufacturer (Life Technologies Corporation). In lieu of this, traditional discontinuous Tris-glycine SDS-PAGE can be carried out using standard methods (Rosenberg, 2005) with comparable results.

Acknowledgments

We thank Professors Michael P. Rout and Torben Heick Jensen for their invaluable support of our research. We also thank Ms. Hua Jiang and Ms. Leila Saba for copyediting. This work was supported in part by the National Institutes of Health grants P41GM109824 and P50GM107632, the Lundbeck Foundation, and the Danish National Research Foundation.

References

  1. Ahuja, D., Saenz-Robles, M. T. and Pipas, J. M. (2005). SV40 large T antigen targets multiple cellular pathways to elicit cellular transformation. Oncogene 24(52): 7729-7745.
  2. Cristea, I. M. and Chait, B. T. (2011). Conjugation of magnetic beads for immunopurification of protein complexes. Cold Spring Harb Protoc 2011(5): pdb prot5610.
  3. Dai, L., Taylor, M. S., O’Donnell, K. A. and Boeke, J. D. (2012). Poly(A) binding protein C1 is essential for efficient L1 retrotransposition and affects L1 RNP formation. Mol Cell Biol 32(21): 4323-4336.
  4. Domanski, M., Molloy, K., Jiang, H., Chait, B. T., Rout, M. P., Jensen, T. H. and LaCava, J. (2012). Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels. Biotechniques 0(0): 1-6.
  5. Domanski, M., Upla, P., Rice, W. J., Molloy, K. R., Ketaren, N. E., Stokes, D. L., Jensen, T. H., Rout, M. P. and LaCava, J. (2016). Purification and analysis of endogenous human RNA exosome complexes. RNA 22(9): 1467-1475.
  6. Freshney, R. I. (2011). Culture of Animal Cells. 6th edition. John Wiley & Sons.
  7. Hakhverdyan, Z., Domanski, M., Hough, L. E., Oroskar, A. A., Oroskar, A. R., Keegan, S., Dilworth, D. J., Molloy, K. R., Sherman, V., Aitchison, J. D., Fenyo, D., Chait, B. T., Jensen, T. H., Rout, M. P. and LaCava, J. (2015). Rapid, optimized interactomic screening. Nat Methods 12(6): 553-560.
  8. Kilchert, C., Wittmann, S. and Vasiljeva, L. (2016). The regulation and functions of the nuclear RNA exosome complex. Nat Rev Mol Cell Biol 17(4): 227-239.
  9. LaCava, J., Jiang, H. and Rout, M. P. (2016). Protein complex affinity capture from cryomilled mammalian cells. J Vis Exp(118).
  10. LaCava, J., Molloy, K. R., Taylor, M. S., Domanski, M., Chait, B. T. and Rout, M. P. (2015). Affinity proteomics to study endogenous protein complexes: pointers, pitfalls, preferences and perspectives. Biotechniques 58(3): 103-119.
  11. Life Technologies Corporation. NuPAGE® Technical Guide. 3188 (Ed.). Life Technologies Corporation.
  12. Muller, N., Girard, P., Hacker, D. L., Jordan, M. and Wurm, F. M. (2005). Orbital shaker technology for the cultivation of mammalian cells in suspension. Biotechnol Bioeng 89(4): 400-406.
  13. Rosenberg, I. M. (2005). Protein analysis and purification. 2nd edition. Birkhäuser Boston.
  14. Taylor, M. S., LaCava, J., Dai, L., Mita, P., Burns, K. H., Rout, M. P. and Boeke, J. D. (2016). Characterization of L1-ribonucleoprotein particles. Methods Mol Biol 1400: 311-338.
  15. Taylor, M. S., LaCava, J., Mita, P., Molloy, K. R., Huang, C. R., Li, D., Adney, E. M., Jiang, H., Burns, K. H., Chait, B. T., Rout, M. P., Boeke, J. D. and Dai, L. (2013). Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition. Cell 155(5): 1034-1048.
  16. Uphoff, C. C. and Drexler, H. G. (2013). Basic Cell Culture Protocols. 4th edition. In: Helgason, C. D. and Miller, C. L. (Eds.). Humana Press.

简介

RNA外植体复合物在RNA加工和调节营养中起核心作用。在细胞质和细胞核中存在,外来体通过与核糖核酸酶和各种衔接蛋白的关联起作用(参见[Kilchert等人,2016])。以下方案描述了从HEK-293细胞中纯化RNA外来体复合物的方法,利用可诱导的异位表达,亲和力捕获和速率 - 区带离心。所获得的RNA外来体已被成功地用于蛋白质组学,结构和酶学研究(Domanski等人,2016)。

在我们以前的工作中,我们建立了在四环素诱导型CMV启动子(HEK-293 Flp-In T-REx-Thermo Fisher)的控制下表达C末端3xFLAG标记的外来体组分EXOSC10(RRP6)的同基因HEK-293细胞系科学)。该系统允许我们以与内源WT蛋白质相当的水平表达标记的EXOSC10蛋白质,并且使用磁性抗FLAG亲和介质和来源于冷冻细胞粉末的蛋白质提取物来研究外来体纯化方案(Domanski等, / em>。,2012)。进一步探索使用的蛋白质提取条件,我们开发了一种允许在亲和捕获的外来体内保留DIS3(RRP44)的方案,否则证明是困难的(Hakhverdyan等人,2015)。基于这些研究,我们通过使用甘油密度梯度的速率区带离心法进一步纯化了RNA外来体(+/- DIS3)(Domanski等人,2016)。虽然蛋白质提取物中洗涤剂CHAPS(3 - [(3-胆酰胺丙基)二甲基铵]丙烷磺酸盐的存在)增加了用亲和捕获的外来体共同纯化的DIS3的产率,但随后在甘油沉降中相互作用丧失密度梯度。为了抵消这一点,使用交联剂DTSSP [3,3'-二硫代双(磺基琥珀酰亚胺基丙酸酯)]。该处理使得在沉降期间保留DIS3在外来体内,但是对DIS3酶功能的负面影响。来自DIS3 +/-外来子级分的峰分数均含有与EXOSC10衍生的分布式3'-5'水解一致的表观核糖核酸裂解活性。保留DIS3(DTSSP处理)的样品和不能通过阴性染色电子显微镜观察到的样品之间的表观结构差异。这里提出的方案将使用户能够获得适合额外分析方法的内源组装的RNA外来子级分,包括体外生物化学,酶学和电子显微镜。注意,该协议的许多方面可以容易地适应,例如使用(1)不同的亲和标签和表达上下文,或(2)抗内源蛋白的抗体(LaCava等人,2015)。

关键字:RNA外泌体, EXOSC10, 低温球磨, HEK-293悬浮培养, 亲和捕获, 速率区带离心

材料和试剂

注意:以下列出的大多数试剂都提供了目录号。来自替代供应商的等效质量试剂通常可以用可比较的结果代替。哺乳动物细胞培养的标准材料和试剂是必需的,下文并没有全部明确列出

  1. 移液器提示
  2. Nunclon细胞培养物245毫米(500厘米2)平皿(Sigma-Aldrich,目录号:D8679)
  3. Nunclon 175cm 2细胞培养瓶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:156502)
  4. 15"细胞刮刀(Fisher Scientific,目录号:08-100-242)
  5. 50ml聚丙烯锥形管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:339652)
  6. 20 ml Luer-lock注射器(BD,目录号:302830)
  7. 石蜡膜(Sigma-Aldrich,目录号:P7793)
  8. 注射器端盖(Bio-Rad Laboratories,目录号:7311660EDU)
  9. 2ml微量离心管(例如,Eppendorf,目录号:022363344)
  10. 0.45μm聚醚砜(PES)无菌注射器过滤器(VWR,目录号:28145-505)
  11. 5 ml超离心管(Seton Scientific,目录号:7022)
    注意:如果您将使用BioComp Instruments活塞梯度分离器来回收纯化外来体的部分(见设备),我们建议您从BioComp获得这些管,因为它们是耐受性测试的兼容性。
  12. HEK-293 Flp-In T-REx EXOSC10-3xFLAG细胞([Domanski等人,2016];可应要求提供)
  13. DMEM,高葡萄糖,GlutaMAX(Thermo Fisher Scientific,目录号:31966047)
  14. 胎牛血清(FBS),四环素免费
    注意:许多供应商可以提供这一点。许多供应商携带没有标记为四环素的FBS产品,但是查询给定批次的产品规格表可能会显示四环素已被测试发现不存在。在我们手中,这些批次的表现与"认证的"无四环素的FBS相同。
  15. 100x青霉素 - 链霉素(P/S)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  16. 胰蛋白酶-EDTA,0.05%(Thermo Fisher Scientific,Gibco TM,目录号:25300054)
  17. 四环素(Sigma-Aldrich,目录号:87128)
    注意:准备10毫克/毫升的乙醇溶液,储存在-20°C。工作溶液为5μg/ml,用于每1ml细胞培养基的诱导加入1μl。也可以使用强力霉素。
  18. 磷酸盐缓冲盐水(PBS),pH 7.4(Thermo Fisher Scientific,目录号:10010023)
  19. 100x L-谷氨酰胺(200mM)(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
  20. TrypLE解离试剂(Thermo Fisher Scientific,Gibco TM,目录号:12605010)
  21. 酚红溶液,0.5%(Sigma-Aldrich,目录号:P0290)
  22. 液氮(LN2)
  23. 自由式293培养基(Thermo Fisher Scientific,Gibco TM ,目录号:12338026)
  24. 3M硫酸铵在0.1M磷酸钠缓冲液pH7.4中
  25. 牛血清白蛋白(BSA)(New England Biolabs,目录号:B9000S)
  26. 甘油(Sigma-Aldrich,目录号:G5516)
  27. 2- [4-(2-羟乙基)哌嗪-1-基]乙磺酸(HEPES)(Sigma-Aldrich,目录号:54457)
  28. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  29. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:71687)
  30. 盐酸(HCl)(Sigma-Aldrich,目录号:320331)
  31. Triton X-100(Sigma-Aldrich,目录号:T8787)
  32. 蛋白酶抑制剂混合物,不含EDTA(Roche Diagnostics,目录号:11873580001)
  33. CHAPS(Sigma-Aldrich,目录号:C5070)
  34. 抗FLAG M2抗体(Sigma-Aldrich,目录号:F3165或F1804)
  35. Dynabeads M-270环氧树脂(Thermo Fisher Scientific,Invitrogen TM,目录号:14302D)
  36. 0.1M磷酸钠缓冲液pH 7.4
  37. Tris碱(Sigma-Aldrich,目录号:93362)
  38. 在TBS(50mM Tris-HCl pH7.4,150mM NaCl)中以5mg/ml重构3xFLAG肽(Sigma-Aldrich,目录号:F4799)。等分试样应储存于-20°C
  39. DTSSP(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:21578)
  40. 4x LDS(十二烷基硫酸钠)样品加载缓冲液(Thermo Fisher Scientific,Novex TM,目录号:NP0007)
  41. 10倍样品还原剂(Thermo Fisher Scientific,Novex TM,目录号:NP0004);或500mM二硫苏糖醇(DTT)
  42. 4-12%Bis-Tris 26孔midi凝胶(Thermo Fisher Scientific,Invitrogen TM,目录号:WG1403BOX)
  43. 20x MOPS(3-吗啉代丙烷-1-磺酸)凝胶运行缓冲液(Thermo Fisher Scientific,Novex TM,目录号:NP0001)
  44. SilverQuest银染色试剂盒(Thermo Fisher Scientific,Novex TM ,目录号:LC6070)
  45. Sypro红宝石蛋白凝胶染色剂(Sigma-Aldrich,目录号:S4942)
  46. 抗EXOSC10抗体(Abcam,目录号:ab95028)
  47. 抗SKIV2L2抗体(Abcam,目录号:ab70552)
  48. 抗-EXOSC3抗体(Proteintech,目录号:15062-1-AP)
  49. ExoI提取溶液(参见食谱)
  50. ExoII提取溶液(参见食谱)
  51. 渐变解决方案 - "轻"(见配方)
  52. 渐变解决方案 - "沉重"(见配方)

设备

注意:以下列出的大多数设备都提供了目录号码。来自替代制造商的仪器可能被替代提供等效的功能

  1. 移液器
  2. 平衡
  3. 用于哺乳动物细胞培养的CO 2培养箱
  4. 冷冻微量离心机(能够达到20,000 x g )
  5. 新不伦瑞克Innova 2000平台摇床(Eppendorf,New Brunswick TM ,型号:Innova ® 2000,目录号:M1190-0002)
    注意:安装在哺乳动物细胞培养箱中的振荡器必须能够耐受连续高湿度(〜90%相对湿度)。
  6. 250毫升塑料烧杯
  7. 血细胞计数器
  8. 1L方形PYREX瓶(Corning,PYREX ®,目录号:1396-1L)
  9. 铣削球,不锈钢,20毫米(Retsch,目录号:05.368.0062)
  10. 50ml不锈钢研磨罐(Retsch,目录号:01.462.0149)
  11. 金属铲子
  12. 行星式球磨机PM 100(Retsch,型号:PM100,目录号:20.540.0001)
  13. Thermomixer(Eppendorf,型号:Thermomixer R,目录号:5355000.011;或等效物)
  14. 装有头部用于多个1.5/2.0ml管的涡旋混合器(Thermo Fisher Scientific,Fisher Scientific,型号:Fisher Scientific TM,Vortex Mixer,目录号:02-215-386)
  15. 钕磁铁微型管架(Thermo Fisher Scientific,目录号:12321D)
  16. 超级离心机与以下列出的转子(Beckman Coulter),例如Optima L或Optima MAX系列(Beckman Coulter,型号:Optima L或Optima MAX系列)兼容。
  17. SW 55 Ti或MLS-50转子(Beckman Coulter,型号:SW 55 Ti或MLS-50)
  18. 装备有低强度1/16"微尖头探针(Qsonica,目录号:4417)的Microtip超声波仪器(例如,Qsonica,型号:Q700)
  19. 梯度分馏器及配件,适用于SW 55 Ti(BioComp Instruments,目录号:152-001)
  20. SW 55 Ti(BioComp Instruments,目录号:107-201M)的梯度主和附件

程序

注意:下面描述的许多步骤用于收获细胞,研磨细胞并进行亲和力捕获,可以在我们的在线视频协议(LaCava等,2016)中查看。

  1. 细胞培养和收获
    可以通过联系作者或按照制造商的说明书获得四环素诱导型EXOSC10-3xFLAG HEK-293细胞系。除非另有说明,适用标准的哺乳动物细胞培养程序(Freshney 2011; Uphoff和Drexler,2013)。
    1. 粘附细胞生长
      1. 在补充有10%v/v FBS和1×P/S(DMEM-FBS)的90ml DMEM中的每个16×500cm 2平方盘上的种子〜10 -P/S) 注意:16 x 500 cm 2 方形盘应产生至少10 g细胞沉淀,湿细胞重量(WCW)。
      2. 当细胞达到〜90%融合时,通过添加补充有5ng/ml四环素(Tet)的新鲜DMEM-FBS-P/S,去除培养基并诱导EXOSC10-3xFLAG的表达。
      3. 通过在冰冷的PBS中刮擦,在诱导后24小时收获细胞 - 我们使用30ml /平板。
        注意:或者,可以通过胰蛋白酶消化来收获细胞。对于每个板,用20ml PBS洗涤细胞,加入10ml的0.05%胰蛋白酶-EDTA,倾斜培养皿以暴露细胞单层,除去多余的液体,并将它们放回到CO 2 孵化器〜5分钟。将细胞重悬于10ml DMEM-FBS-P/S中并转移到50ml管中。
      4. 将收获的细胞以1,000×g离心5分钟(4℃)
      5. 丢弃上清液,重新悬浮,并将沉淀物置于冰冷的PBS(约100ml总体积)中,在两个50ml管内。
      6. 以1,000×g离心5分钟(4℃)。
      7. 丢弃上清液,将沉淀物重新悬浮在冰冷的PBS(每体积小瓶中约1体积的PBS)中,并将溶液转移到20ml的封盖注射器中。用Parafilm密封注射器的上部开口(以避免任何意外溢出),并将其置于50ml管内。
      8. 以1,000 x g离心5分钟(4°C)
      9. 取出任何剩余的液体,更换柱塞,并小心地将内容物注入LN2。
        注意:首先,用LN2将250ml塑料烧杯(放置在聚苯乙烯泡罩内),然后将细胞直接注入LN2。
      10. 储存于-80°C,然后冷冻。
    2. 悬浮细胞生长
      对于该协议,您将需要一个具有适当公差的平台振动器,安装在37°C,加湿8%CO 2培养箱内。悬浮细胞生长为生成大量细胞并保持连续培养提供了便利且具有成本效益的方法。本文给出的方案是根据我们以前调整的方法(Muller等人,2005)改编的,并应用于HEK-293T LD细胞(Dai等人,/em>,2012; Taylor等人,2013; Taylor等人,2016)。使用HEK-293 Flp-In T-REx细胞,悬浮液生长可能产生约3-5万个细胞/ml,生长减缓至约300万/ml。获得准确的细胞计数可能是具有挑战性的,因为这些悬浮液倾向于以块状生长,并且需要通过移液进行研磨以在血细胞计数器中计数。 WCW的产量应在接近内源水平时每400ml表达3xFLAG标记的EXOSC10的细胞培养物≥4g。
      1. 种子细胞以标准方式从股票获得,并获得接近汇合(90%+)细胞的2×175cm 2烧瓶。我们使用25ml培养基/175cm 2烧瓶。
      2. 将DMEM-FBS-P/S和补充有2%v/v FBS,2mM L-谷氨酰胺的Freestyle培养基和1:1混合物中的细胞分成4×175cm 2烧瓶0.5x P/S(后者,称为调理介质)。这启动了粘附细胞的调理以用于悬浮生长。
        注意:我们不能直接从冷冻细胞悬浮液调理的HEK-293 Flp-In T-REx细胞接种新的悬浮培养物。因此,我们为每个新的悬浮液生长新鲜调节细胞,然后将细胞维持在所需持续时间的悬浮液中。与HEK-293T LD细胞相比,HEK-293 Flp-In T-REx细胞观察到的直接接种到悬浮液的难度和较低的最大细胞密度可能是由于SV40大T前者的抗原(Ahuja et al。,2005; Taylor et al。,2016)。
      3. 在下一次细胞分裂时,使用DMEM-FBS-P/S和调理培养基的1:3混合物,将细胞转移到8×175cm 2烧瓶中,并生长至接近汇合。
      4. 用10ml PBS洗涤细胞并丢弃洗涤液,然后使用0.1x TrypLE(PBS中1:10稀释)从烧瓶中释放细胞。加入1.5ml 0.1x TrypLE溶液,倾斜每个烧瓶暴露细胞单层,然后除去多余的液体。将烧瓶在室温下孵育10分钟
      5. 将释放的细胞从每个烧瓶中重悬在10ml的调理培养基中。将40ml细胞悬浮液和60ml补充有200μl0.5%w/v酚红(最终0.001%w/v)的调理培养基合并在1L方形玻璃瓶中。这通常导致两个100 ml悬浮培养物,细胞密度约为每毫升几百万。悬液培养物应接种在0.5-300万个细胞/ml之间。离开瓶盖一整圈打开(从刚刚关闭),并将孵化器内的文化摇摆速度为130 RPM。
        注意:酚红(PR)作为pH指示剂和监测培养细胞密度的间接方法,补充细胞计数。该溶液应具有类似于市售DMEM的浅红色外观,其也经常含有该添加剂。该协议中1升玻璃瓶的工作容积在100-400毫升之间。两个400毫升培养物最终应产生至少8克WCW。
      6. 通过细胞计数和中等颜色监测文化。当细胞密度非常接近或超过300万个细胞/ml并开始变成橙色时,将培养物稀释至1-2百万个细胞/ml,其中加入补充有2mM L-谷氨酰胺的0.5ml Freestyle培养基,0.5x P/S和0.001%PR(无FBS) - 在1%最终FBS浓度下进行,而其他试剂浓度保持不变(称为悬浮培养基)。所有未来的稀释液都可以用悬浮液进行。
        注意:细胞通常是圆形的,单一的,并且在初始接种后几天相对易于计数,然后将变得越来越团块化,甚至在一定程度上粘附到液体线到达的烧瓶侧。
      7. 通过细胞计数和中等颜色继续监测培养。当细胞密度非常接近或超过300万个细胞/ml并开始变成橙色时,用200ml悬浮培养基将培养物稀释至≤300万个细胞/ml。
      8. 一旦这400毫升的培养基非常接近或超过300万个细胞/毫升,从每个培养基中除去50毫升的培养物,并使用它来以约0.5-2百万个细胞/ml(如果需要)种两个或更多个新的100ml培养物, 。向剩余的350ml培养物中加入50ml补充有8ng/ml Tet(终浓度为1ng/ml)的悬浮培养基,孵育过夜(〜16-24小时)。
        注意:我们观察到悬浮培养物中1ng/ml的四环素足以使贴壁生长中相当于5ng/ml的表达。然而,我们鼓励研究人员通过在诱导后进行抗RRP6蛋白质印迹来测试手中的表达(Domanski等,2012)。该方案可以使用较小的培养容器进行小型化,以进行这种性质的预测试。
      9. 通过离心分离细胞,在LN 2中洗涤和冷冻,如A1部分中的步骤d-j所述 - 粘附细胞生长 -

  2. 冷冻程序
    之前已经描述了哺乳动物细胞沉淀的低温破坏(Domanski等人,2012; Taylor等人,2016; LaCava等人/em >。,2016)。简而言之,通过浸入LN 2中预先冷却研磨罐,2×20mm研磨球,金属刮刀和50ml管。从罐室中取出任何LN 2 ,并将细胞颗粒放入其中。设置计数器平衡并将罐子夹紧在Retsch PM 100中。运行机器3轮铣削循环,每次3分钟(反向旋转,1分钟间隔,无断裂时间),转速为400 RPM。在循环之间用LN 2 2冷却研磨罐。使用刮刀回收所得细胞粉末并转移到50ml管中。储存于-80°C。

  3. 抗FLAG M2抗体与Dynabeads M-270环氧树脂偶联
    之前已经详细描述了抗FLAG磁性介质的生产(Cristea和Chait,2011; Domanski等人,2012; Taylor等人,2016)和也可以根据制造商的说明(Thermo Fisher Scientific)进行,具有可比的结果。简言之,在磷酸钠(pH 7.4)缓冲1M硫酸铵溶液中制备20μl0.5μg/μl抗体,每mg磁珠以抗体缀合(即,10μg的抗体,在20μl溶液中,将被使用/mg珠)。结合抗体溶液和磁珠,并在30-37℃下旋转孵育过夜(16-24小时)。所施加的混合物应足以确保珠子在孵育期间保持悬浮状态。对于小批量,可以在37℃的温热混合器(≥1200RPM)中进行共轭。偶联后,除去过量的抗体溶液,洗涤珠子以除去残余的未偶联的抗体,最后将抗体缀合的磁性介质重悬于含有0.5mg/ml BSA和50%v/v甘油(储存溶液)的1×PBS(最终) :每1 mg培养基添加6.7μl储存溶液(产生约15%w/v的浆液)并储存于-20°C。浆料可以以这种方式储存至少1年,而没有明显的性能损失。

  4. 亲和纯化
    亲和纯化
    以前已经描述了描述和证明使用哺乳动物细胞粉末和磁性介质的亲和纯化的最佳实践的一般程序(LaCava等人,2016)。以下实施已经被优化用于获得人类RNA外来体。这里,DIS3-外来体被称为ExoI,DIS3 +外来体是ExoII。
    1. 在LN 2中预冷金属刮刀和4 x 2 ml安全锁管。
    2. 重量4×250毫克HEK-293 EXOSC10-3xFLAG细胞粉末。
      注意:保持细胞粉末冻结非常重要。将管留在LN2中直到下一步启动。
    3. 将样品放在机架上,打开盖子,让它们在室温下放置1分钟。
    4. 向每个添加1,250μlExoI或ExoII解决方案(请参阅配方)。
      注意:ExoI溶液产生DIS3-外来体,而ExoII产生DIS3 +外来体。
    5. 以最大速度涡旋以完全重悬细胞粉(不应超过30秒),并立即将样品置于冰上。
      注意:一旦重新悬浮细胞粉末,除非另有说明,样品应在整个操作过程中的所有操作之间保持在冰上。
    6. 简单地超声处理以完全分散和均化细胞粉末。使用4个脉冲,每次2秒(每250mg样品约30 J总量)(LaCava等人,2016)。
      注意:如果结块可见,应重复超声处理步骤,直到通过目视检查才能显示出明显的结块。
    7. 通过以20,000×g离心10分钟(4℃)澄清提取物。
    8. 当样品在离心机中时,将4x 25μl抗FLAG珠粒浆液分配到2 ml微量离心管中,并用1 ml提取溶液洗涤两次,彻底清除存储溶液。
      注意:我们每100毫升细胞粉末使用10微升的珠浆。为了洗涤珠子,将1ml提取溶液与25μl抗FLAG磁性介质(浆液)混合在2ml微量离心管中,短暂旋转以完全重悬珠。在微型离心机中短暂地旋转管子以收集底部的所有溶液,然后将管放置在磁性管架中,直到在管的侧面收集珠粒。使用移液管或吸液器除去上清液,并重复洗涤步骤一次。在室温下进行两个洗涤步骤。洗涤后,珠可以保持在冰上并准备使用。或者,也可以洗涤总共100μl抗FLAG培养基,并在洗涤后分布在4管中。
    9. 将澄清的提取物与预洗珠组合。孵育1 h,旋转4°C(冷室)。
    10. 将磁珠收集在磁体上,取出上清液,并用上述方法用1ml冰冷的提取溶液洗涤。
    11. 用提取液再洗两次。在第二次洗涤期间将每个样品移动到新鲜管中 注意:将每个样品(珠子重新悬浮在提取溶液中)移至新鲜管中,可最大限度地减少洗脱液的污染。非特异性吸附到用于亲和力捕获的管的内表面的细胞提取物蛋白可以在随后的操作期间释放到样品中。
    12. 在3小时以上洗涤后,在小型离心机中旋转样品。
    13. 再次放在磁铁上,小心地取出剩余的液体。
    14. 向每个管中加入30μl洗脱溶液(1μg/ml 3xFLAG肽,从提取溶液中的浆液中稀释)。
    15. 在室温下温和搅拌15分钟。 〜60%功率的涡流装备有多个1.5/2.0 ml管的头。
      注意:确保珠子不会过度混合,也不会沉入管子的底部。
    16. 放在磁铁上并收集上清液。
    17. 加入30μl提取液洗涤珠粒。收集如上所述,将分数汇集在一起。
    18. 将样品放在冰上,然后加载到梯度上。

  5. DTSSP交联获得ExoII
    在ExoII提取溶液中,DIS3可以与外来体共同纯化。然而,它在沉淀过程中以甘油梯度分解。为了保持这种相互作用,我们使用了化学交联。 DTSSP将增强DIS3/exosome相互作用的保存,但DIS3酶活性将受到这种治疗的影响。
    1. 加入2μl新鲜制备的1.2mM DTSSP /10μlExoII,通过如上所述用3xFLAG洗脱而制备。
    2. 在室温下进行交联反应50分钟。
    3. 通过加入2μl1M Tris-HCl pH8淬灭
    4. 在加载梯度之前保持在冰上。

  6. 率区域离心力
    Beckman Optima L系列超速离心机中的SW 55 Ti转子的以下设置和参数进行了优化。有关MLS-50的详细信息,请参阅(Domanski等人,2016年)。
    1. 使用BioComp Gradient Master仪器(短帽,10-40%甘油v/v)或其他合适的方法制备10-40%甘油梯度。始终准备一个额外的渐变以用作平衡。
    2. 预冷形成梯度至4℃(在冷室中典型地≥1小时)。
    3. 通过轻轻移液将样品加载到梯度的顶部。
      注意:为了避免破坏梯度的顶层,将移液管尖端靠在管壁上,恰好在甘油溶液的半月板上方,并缓慢地喷出样品。
    4. 使用以下设置运行渐变:最小加速度;无刹车4℃; 50k RPM; 6小时36分。
    5. 运行完成后,使用BioComp活塞梯度分馏器(参见制造商的说明书)或其他适当的方法收集馏分。我们使用手动分数收集的以下设置:distance-2 mm,speed-0.3 mm/sec。该收集方案将产生半月板分数,〜20个分数为〜225μl,底部馏分。根据实施的具体设置,将观察到一些变化,然而,使用所描述的程序和设置,我们检索了分数11和12中的分数11和ExoII中沉积的ExoI的峰值。

  7. 梯度分数的SDS-PAGE分析
    1. 用5μl4x LDS和1μl10x还原剂(或500 mM DTT)混合14μl梯度馏分;对于ExoI(DIS3-)级分,在70℃下加热10分钟,或对于DTSSP处理的ExoII(DIS3 +)馏分在75℃下加热20分钟。
    2. 按照制造商的说明书将样品装入26孔,4-12%NuPAGE Bis-Tris凝胶上
    3. 以200 V运行,直到跟踪染料到达凝胶盒底部。
    4. 从塑料盒中取出凝胶,并放入干净的容器中
    5. 按照制造商的说明,用银或Sypro Ruby染色。

数据分析

在沉淀期间,游离蛋白质(或部分组装的复合物)可以靠近梯度顶部发现,而完整的RNA外来体进一步迁移并且可以在梯度中间附近发现。含有完整外来体的级分表现出(来自凝胶顶部)SKIV2L2,EXOSC10-3xFLAG,EXOSC9和核心/低质量蛋白质的标志性染色强度,大约分布在预期分子质量(EXOSC9比其预测质量更接近60kDa) 〜49 kDa)。图1A描述了经超速离心后获得的梯度级分(图1B中所示的真实数据)的SDS-聚丙烯酰胺凝胶染色。在这个例子中,分数11(11)表明组成和染色强度与峰值分数一致。存在于梯度级分中的蛋白质的浓度应足以通过例如银或Sypro Ruby染色直接检测。蛋白质鉴定可以通过使用特异性抗体的Western印迹(参见注释部分)和/或质谱来确认。


图1.代表性的结果:从表达EXOSC10-3xFLAG的HEK-293细胞纯化的RNA外来体。A.染色的SDS-聚丙烯酰胺凝胶的示意图,证明与分离EXOSC10-3xFLAG纯化的RNA一致的蛋白条带在10-40%v/v甘油梯度内沉降后获得的外来体。 B.由Domanski等人于2016年出版的原始凝胶。分离的蛋白质通过银染色显现。箭头表示峰值分数。

笔记

列出了一些商业上可获得的抗体,我们成功地通过Western印迹鉴定外来体成分。

  1. 抗EXOSC10抗体(Abcam,目录号:ab95028)
  2. 抗SKIV2L2抗体(Abcam,目录号:ab70552)
  3. 抗-EXOSC3抗体(Proteintech,目录号:15062-1-AP)

食谱

  1. ExoI提取溶液
    20mM HEPES-NaOH,pH 7.4
    300 mM NaCl
    1%Triton X-100(v/v)
    1x蛋白酶抑制剂鸡尾酒
  2. ExoII提取溶液
    20mM HEPES-NaOH,pH 7.4
    100 mM NaCl
    5 mM CHAPS
    1x蛋白酶抑制剂鸡尾酒
  3. 1M Tris-HCl,pH 8.0(用于淬灭DTSSP交联反应)
  4. 渐变解决方案 - "轻"
    10%v/v甘油
    20mM HEPES-NaOH,pH 7.4
    100 mM NaCl
    带0.45μm注射器过滤器的无菌过滤器
  5. 渐变解决方案 - "沉重"
    40%甘油
    20mM HEPES-NaOH,pH 7.4
    100 mM NaCl
    带0.45μm注射器过滤器的无菌过滤器

注意:NuPAGE ®系统中使用的许多解决方案可以在实验室进行,不需要购买。请咨询制造商提供的食谱(Life Technologies Corporation)。代替这一点,传统的不连续的Tris-甘氨酸SDS-PAGE可以使用标准方法(Rosenberg,2005)进行,具有可比较的结果。

致谢

感谢Michael P. Rout教授和Torben Heick Jensen教授对我们研究的宝贵支持。我们也感谢华江女士和雷拉·萨巴女士的拷贝。这项工作得到了美国国家卫生研究院授予的P41GM109824和P50GM107632,Lundbeck基金会和丹麦国家研究基金会的支持。

参考文献

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  9. LaCava,J.,Jiang,H.and Rout,MP(2016)。  来自低温哺乳动物细胞的蛋白质复合物亲和力捕获 J Vis Exp (118)。
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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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