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Recently, we identified two host cell-derived proteins as novel stimulatory factors of influenza virus RNA replication process, termed “Influenza virus REplication Factor-2 (IREF-2)”, from human nuclear extracts (NEs) by employing biochemical complementation assays (Sugiyama et al., 2015). Herein, we describe detailed methods for successive procedures for identification and purification of IREF-2, including large-scale suspension culture of HeLa S3 cells, preparation of NEs and separation of IREF-2 by sequential column chromatography steps. This protocol can be modified and used for purification and identification of the other unknown nuclear protein(s) of your interest.

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Purification and Identification of Novel Host-derived Factors for Influenza Virus Replication from Human Nuclear Extracts
从人细胞核提取物纯化和识别用于流感病毒复制的新的宿主源因子

微生物学 > 微生物-宿主相互作用 > 体外实验模型
作者: Kenji Sugiyama
Kenji SugiyamaAffiliation: Department of Infection Biology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
For correspondence: kenjisugiyamascience@gmail.com
Bio-protocol author page: a3516
 and Kyosuke Nagata
Kyosuke NagataAffiliation: Department of Infection Biology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
Bio-protocol author page: a3517
Vol 6, Iss 18, 9/20/2016, 1422 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1934

[Abstract] Recently, we identified two host cell-derived proteins as novel stimulatory factors of influenza virus RNA replication process, termed “Influenza virus REplication Factor-2 (IREF-2)”, from human nuclear extracts (NEs) by employing biochemical complementation assays (Sugiyama et al., 2015). Herein, we describe detailed methods for successive procedures for identification and purification of IREF-2, including large-scale suspension culture of HeLa S3 cells, preparation of NEs and separation of IREF-2 by sequential column chromatography steps. This protocol can be modified and used for purification and identification of the other unknown nuclear protein(s) of your interest.
Keywords: Nuclear Extracts(核提取物), Purification and identification(纯化与鉴定), Influenza(流感), Host factor(宿主因素), IREF-2(iref-2)

[Background] The influenza A virus genome is composed of 8 segmented- and single stranded-RNA (vRNA). Its transcription and replication are catalyzed by virus-encoded RNA-dependent RNA polymerase (RdRP). Several lines of evidence suggest that certain host-derived factors regulate the viral RNA syntheses (Nagata et al., 2008). Recently, a variety of host-derived proteins have been reported as regulator candidates related to the viral RNA syntheses by interactome analyses and genome wide RNAi screening studies. Among them, however, some false-positive interactors and factors involved indirectly in the viral RNA synthesis seem to be included. Instead, to identify reliable and important host factor(s) which play a direct role in the viral RNA synthesis process, we utilized a biochemical complementation assay system. In this system, the viral vRNA replication reaction occurring efficiently in infected cell nuclei is dissected and reconstituted in vitro using viral factors essential for the viral RNA replication such as viral RdRP derived from detergent-solubilized virion particle and model viral genomic RNA templates and uninfected nuclear extracts (NEs).
  Recently, we have reported that an efficient vRNA replication is reproduced in vitro with viral factors and a crude fraction of NEs (Sugiyama et al., 2015). This stimulatory activity presented in NEs, designated IREF-2 that allows robust vRNA replication, was further fractionated and purified by sequential column chromatography. Finally, two host-derived paralogous proteins, pp32 (accession number NP_006296) and APRIL (accession number NP_006392), were identified by MS spectrometry analyses. Following experiments using recombinant pp32 and APRIL, and also in vivo analyses using siRNA confirmed that these host proteins are authentic proteins responsible for the IREF-2 activity (Sugiyama et al., 2015).

[Abstract]

Materials and Reagents

  1. Tissue culture dish
  2. Centrifuge tubes [e.g., for F0650 rotor (Beckman Coulter, catalog number: 363075 )]
  3. Conical centrifuge tubes (polypropylene, 50 ml) (e.g., Corning, catalog number: 430829 )
  4. Conical centrifuge tubes (polypropylene, 15 ml) (e.g., Corning, catalog number: 430791 )
  5. Dialysis membrane tube (Cellulose membrane, 14,000 dalton molecular weight cut off), (EIDIA, catalog number: UC27-32-100 )
  6. Bottle top filter (Merck Millipore Corporation, catalog number: SCGPS01RE )
  7. Microcentrifuge tubes (polypropylene, 1.5 ml size and 0.6 ml size) (e.g., Eppendorf)
  8. HeLa S3 cell [National Institutes of Biomedical Innovation, Japanese Collection of Research Bioresources (JCRB), catalog number: JCRB0713]
  9. Minimum essential medium Eagle (MEM) (Sigma-Aldrich, catalog number: M4655 )
  10. Fatal bovine serum (FBS) (e.g., Thermo Fisher Scientific, GibcoTM, catalog number: 26140-079 )
  11. S-MEM [Sigma-Aldrich, catalog number: M4767 (production ceased, see Note 1). Alternatively, see also Sigma-Aldrich, catalog number: M8167 , or similar products from other companies]
  12. Bovine serum (CS) (e.g., Thermo Fisher Scientific, GibcoTM, catalog number: 16170-078 )
  13. Ultra-pure water (Milli-Q)
  14. Phosphate buffered saline (PBS)
  15. 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) buffer
  16. Sodium hydroxide (NaOH)
  17. Hydrochloric acid (HCl)
  18. Sodium chloride (NaCl)
  19. Potassium chloride (KCl)
  20. Magnesium chloride hexahydrate (MgCl2)
  21. Dithiothreitol (DTT)
  22. Trypsin-EDTA (for cell trypsinization)
  23. Glycerol
  24. di-Sodium dihydrogen ethylendiamine tetraacetate dihydrate (EDTA)
  25. Phenylmethylsulfonyl fluoride (PMSF)
  26. Dimethyl sulfoxide (DMSO)
  27. Liquid N2
  28. Whatman® cation exchange cellulose (P11) (Sigma-Aldrich, catalog number: 4071010 )
  29. Ammonium sulfate [(NH4)2SO4]
  30. Silver staining kit (e.g., Cosmo Bio, catalog number: DCB-423413 )
  31. Silver staining kit (grutalaldehyde-free for MS analysis) (e.g., Wako Pure Chemical industries, catalog number: 299-58901 )
  32. Buffer A (see Recipes)
  33. Buffer C (see Recipes)
  34. Buffer D (see Recipes)
  35. Buffer HG (see Recipes)
  36. Buffer HGK (see Recipes)
  37. Buffer HGN1M (see Recipes)
  38. Buffer HGN2M (see Recipes)

Equipment

  1. Cell culture environments and equipments
    1. Clean bench
    2. CO2 incubator (37 °C for adherent cultivation)
    3. Microscope (phase-contrast)
    4. Hemocytometer
    5. Spinner flasks (small size for ~500 ml and large size for 8 L)
    6. Magnetic stirrer (Slow speed; enough size for spinner flask)
    7. 37 °C incubator (enough size for spinner flask and magnetic stirrer)
    8. Autoclave (enough size for spinner flask)
  2. Large scale centrifuge (e.g., Beckman Coulter, model: Avanti® J-26XP )
  3. Large scale centrifuge rotor (Beckman Coulter, model: JLA-10.500 )
  4. Centrifuge bottles (500 ml-size for JLA-10.500 rotor) (e.g., Beckman Coulter, catalog number: 361691 )
  5. Benchtop high speed centrifuge (e.g., Beckman Coulter, model: Allegra® 64R )
  6. High-speed centrifuge rotor [e.g., F0650 (Beckman Coulter, catalog number: 364610 )]
  7. Grass dounce homogenizer with pestle, 15 ml (Capitol Scientific, Wheaton®, catalog number: 357544 )
  8. Grass dounce homogenizer with pestle, 40 ml (Capitol Scientific, Wheaton®, catalog number: 357546 )
  9. Refrigerator (for dialysis)
  10. Deep freezer (-80 °C)
  11. pH meter
  12. Grass bottles (Pyrex®, dry heat-sterilizable)
  13. Tabletop centrifuge
  14. SDS-PAGE apparatus
  15. Column chromatography equipments (see Note 1)
    1. Fast protein liquid chromatography (FPLC) workstation (Bio-Rad Laboratories, model: BioLogic HR )
    2. Chromato chamber (4 °C)
    3. Column housing (e.g., Bio-Rad Laboratories, Econo Column®, catalog number: 7371512 )
    4. Flow adaptor (e.g., Bio-Rad Laboratories, catalog number: 7380016 )
    5. Anion-exchanger, UNOTM Q1R (Bio-Rad Laboratories, catalog number: 7200011 )
    6. Hydrophobic column, SOURCETM 15PHE PE 4.6/100 (GE Healthcare, catalog number: 17-5186-01 )
    7. Cation-exchanger, UNOTM S1 (Bio-Rad Laboratories, catalog number: 7200021 )
    8. Anion-exchanger, Mono Q® HR 5/5 [GE Healthcare, model: Mono Q® HR 5/5 (production ceased). Alternatively, Mono Q® 5/50 GL (GE Healthcare, model: 17-5166-01 ) or similar products from other companies can be usable]
    9. Micro-Purification SMART system [GE Healthcare, Pharmacia Biotech, model: SMART® system (production ceased)]
    10. Gel filtration column (GE Healthcare, Pharmacia Biotech, model: Superdex® 75 PC 3.2/30 )
      Note: This product has been discontinued.
  16. Razor blade

Procedure

  1. Large scale culture of HeLa S3 cells (see Note 2)
    1. Seed HeLa S3 cells on tissue culture dish(es) in MEM + 10% FBS medium.
    2. Incubate at 37 °C in 5% CO2 to grow until total cell number reaches to ≥ 1 x 107. Keep the density of adherent cells within 30-80% confluence. If necessary, passage all of the growing cells by increasing size and/or numbers of culture dish(es).
    3. Trypsinize the cells, resuspend in S-MEM + 5% calf serum (CS), and count the number of cells.
    4. Adjust the cell density to ~2 x 105 cells/ml by adding S-MEM + 5% CS.
    5. Place the cell suspension into a 500 ml-size spinner flask (sterilized), and incubate at 37 °C with gentle stirring by a magnetic stirrer (60-80 rotations/min).
    6. Monitor the cell growth by daily counting the number of cells, and add fresh medium (S-MEM + 5% CS) to maintain cells at 3-6 x 105 cells/ml. When the volume of cell culture reaches to 500 ml, transfer the culture to a large-scale spinner flask (8 L-size) and maintain incubation at 37 °C.
    7. When the cell culture reaching maximum volume (e.g., 8 L of 6 x 105 cells/ml; approximately 5 x 109 of total cells), proceed to the nuclear extracts preparation steps (see Note 3).

  2. Nuclear Extracts (NEs) preparation from HeLa S3 cells (see Note 4)
    1. Harvest the cultured HeLa S3 cells by centrifugation (at 1,500 x g, 4 °C for 10 min).
    2. Suspend the cell pellet in PBS, and collect by centrifugation.
    3. Suspend the cells in ~5 packed cell volumes (PCV) (i.e., pellet mass of the collected cells) of ice-cold buffer A, and store on ice for 10 min (see Note 5).
    4. Collect by centrifugation at 1,500 x g for 10 min at 4 °C.
    5. Suspend the cells in 2 PCV of the buffer A and allow cells to swell on ice for 10 min.
    6. Lyse the cells by 10-15 strokes of a dounce homogenizer with “type B (tight)” pestle.
    7. Check for the cell lysis under a microscopy (Figure 1). If the homogenization appears to be insufficient, homogenize additionally and check the lysis again.


      Figure 1. Microscopic view of the cell homogenate. Suspensions before and after homogenization (step B6) were observed at 200-fold magnification by phase-contrast microscopy. A. Cells suspended in PBS (step B2); B. Swollen cells in a hypotonic “buffer A” before homogenization (step B5); C. Homogenized cell suspension (step B6). Cytoplasmic debris and nuclei were observed.

    8. Centrifuge the homogenate at 3,300 x g for 10 min at 4 °C, and remove the supernatant. The supernatant removed at this step can be saved as “S-100” cytoplasmic extracts (Dignam et al., 1983), and utilized for other purpose (Matsumoto et al., 1993).
    9. Re-centrifuge the remaining pellet (i.e., nuclei) at 14,000 x g for 20 min at 4 °C and remove the residual supernatant completely.
    10. Suspend the cells in equal volume of packed nuclei (i.e., equal to pellet mass of the collected nuclei) with ice-cold buffer C (see Recipes and Note 6) and homogenate by 10 strokes of a dounce homogenizer with “type B (tight)” pestle to ensure complete suspension.
    11. Transfer the homogenate to a centrifuge tube and invert gently by rotating for 30 min at 4 °C.
    12. Centrifuge the homogenate at 25,000 x g for 10 min at 4 °C.
    13. Place the supernatant (NEs) in a dialysis membrane bag. The remaining pellet at this step can be frozen and stored at -80 °C, and utilized as a chromatin fraction (Simon and Felsenfeld, 1979; Schnitzler, 2001).
    14. Dialyze against ≥ 50 volumes of buffer D at 4 °C for 3 h. Change the buffer to fresh buffer D (≥ 50 volumes), and additionally dialyze at 4 °C for 3 h.
    15. Transfer the dialysate (white-turbid NEs) to a centrifuge tube and centrifuge for 20 min at 25,000 x g by benchtop centrifuge.
    16. Determine the protein concentration of NEs of the supernatant (see Note 7). Dispense aliquots into tubes (if desired), and freeze in liquid N2 and store at -80 °C until use (see Note 8).

  3. Fractionation and purification of NEs by successive column chromatographies (see Figure 2 and Note 9). Herein, the procedures of the column chromatography are desired to purify and identify two host-derived factors, pp32 and APRIL (as known as ANP32A and B, respectively), termed as “influenza virus replication factor (IREF)-2” which stimulates influenza virus RNA replication process (Sugiyama et al., 2015).


    Figure 2. Purification scheme of IREF-2 from NEs. Chromatographic behavior of IREF-2 appears to be highly acidic and hydrophilic.

    1. Stepwise fractionation on weak cation-exchanger column
      1. Prepare phosphocellulose (PC) column, as follows:
        1. Suspend the powder of PC resin (Whatman® cation exchange cellulose, P11) by gentle swirling in ≥ 20 volumes of water and keep for 30~60 min.
        2. Pour off supernatant, resuspend the resin in water, and repeat at least 8-10 times to remove any unsettled fine.
        3. Suspend the resin in 0.2 M NaOH, allow to settle for 30 min.
        4. Pour off supernatant and repeat until pH of supernatant is above 10.
        5. Wash the resin with water until pH of supernatant is below 8.
        6. Suspend the resin in 0.2 M HCl, allow to settle.
        7. Pour off supernatant and repeat until pH of supernatant is below 3.
        8. Wash the resin with water until pH of supernatant is about 5.
        9. Equilibrate the resins in buffer HG. If the PC resin is to be stored for longer than a week, add 0.1% sodium azide and store at 4 °C (never freeze).
        10. Suspend the PC resin and pour carefully into the column housing [approximately, 10 ml (bed volume) of the PC resin is enough for 10~15 ml of input NEs] and allow to settle. Then, attach the flow-adaptor carefully above the bed.
      2. Set up the FPLC system and column, routinely as follows:
        1. Wash the system (including a “sample loop”) with pure degassed water.
        2. Connect the prepared PC column (10 ml bed volume) to the system without introducing air, and wash the system including the column with enough water. Ensure no leakage from the column during flow.
        3. Set degassed buffer HG as solvent A to inlet A and degassed buffer HGK as solvent B to inlet B, respectively.
        4. Wash the system with 100% solvent B (buffer HGK; 1 M KCl) and then equilibrate with 5% solvent B (i.e., 95% solvent A of buffer HG; 50 mM KCl).
      3. Ensure the valve position to be “load”, and load the NEs (approximately, 12.5 ml in this protocol) into the sample loop.
      4. Change the valve position to “inject”, and run the program to start stepwise fractionation on the PC column (10 ml bed volume), as follows:
        1. Isocratic flow (0.15 ml/min) with 45 ml (4 volumes of the input NEs) of 5% solvent B (i.e., 95% solvent A; final 0.05 M KCl), collect the eluate (2 ml/fraction; total 23 fractions).
        2. Change the valve position from “inject” to “load” to bypass the flow channel through the sample loop. If forget this, following salt-elution steps would be delayed by the volume of the sample loop.
        3. Isocratic flow (0.15 ml/min) with 40 ml (4 column volumes) of 20% solvent B (i.e., 80% solvent A; final 0.2 M KCl), collect the eluate (2 ml/fraction; total 20 fractions).
        4. Isocratic flow (0.15 ml/min) with 40 ml (4 column volumes) of 50% solvent B (i.e., 50% solvent A; final 0.5 M KCl), collect the eluate (2 ml/fraction; total 20 fractions).
        5. Isocratic flow (0.15 ml/min) with 40 ml (4 column volumes) of 100% solvent B (i.e., 0% solvent A; final 1 M KCl), collect the eluate (2 ml/fraction; total 20 fractions).
      5. Make small aliquot(s) of each fractionated sample to avoid unnecessary freezing/thawing cycle in future, and freeze in liquid N2 and store at -80 °C.
      6. Check the activity (i.e., stimulation activity for influenza virus RNA replication, termed as “IREF-2” in this protocol) involved in the PC-fractions by employing a cell-free viral RNA replication assay using small aliquot (see Figure 3 and Note 10).


        Figure 3. Profile of the fractions from phosphocellulose column chromatography for IREF-2. Each stepwise fraction of NE from the phosphocellulose (PC) column chromatography was individually added to the cell-free viral RNA replication reaction (1.75 μg of each fraction/reaction), as follows; unbound fraction (i.e., flow-through) eluted with 0.05 M KCl buffer (lane 1), materials eluted with buffer containing 0.2 M KCl (lane 2), 0.5 M KCl (lane 3) and 1 M KCl (lane 4), respectively. For the detail of the cell-free viral RNA replication assay, see Note 10 and the original article (Sugiyama et al., 2015). Robust viral RNA product was detected (upper panel, lane 1), suggesting that certain activity stimulating a viral RNA replication reaction, termed as IREF-2, was present in the flow-through fraction from the PC column chromatography. Each fraction was also subjected to 14% SDS-PAGE (1 μg of each fraction/lane), and polypeptides were visualized by silver staining (lower panel). The arrow indicates viral RNA replication products. The molecular weight (kDa) positions are denoted on the right side of the lower panel. MWM: molecular weight maker.

    2. Stepwise fractionation on a strong anion-exchanger column
      1. Set up the FPLC system and pre-packed UNO Q1R column, routinely
        1. Set degassed buffer HG as solvent A to inlet A and degassed buffer HGK as solvent B to inlet B, respectively.
        2. Wash the column with 100% solvent B, and equilibrate with 5% solvent B (i.e., 95% solvent A).
      2. Thaw the IREF-2 activity-positive fraction (total 9 ml in this protocol) of the PC column chromatography (i.e., the unbound “flow-through” fraction, see Figure 3) and centrifuge at 20,000 x g for 5 min at 4 °C to remove insoluble material.
      3. Load the supernatant into the sample loop and change the valve position to “inject”. Run the program to start stepwise fractionation on the UNO-Q column (1.3 ml column volume) as follows:
        1. Isocratic flow (0.5 ml/min) with 18 ml (2 volumes of the input) of 5% solvent B (i.e., 95% solvent A; final 0.05 M KCl), collect the eluate (1 ml/fraction; total 18 fractions).
        2. Change the valve position from “inject” to “load”.
        3. Isocratic flow (0.5 ml/min) with 7.8 ml (6 column volumes) of 15% solvent B (i.e., 85% solvent A; final 0.15 M KCl), collect the eluate (1 ml/fraction; total 8 fractions).
        4. Isocratic flow (0.5 ml/min) with 7.8 ml (6 column volumes) of 30% solvent B (i.e., 70% solvent A; final 0.3 M KCl), collect the eluate (1 ml/fraction; total 8 fractions).
        5. Isocratic flow (0.5 ml/min) with 7.8 ml (6 column volumes) of 60% solvent B (i.e., 40% solvent A; final 0.6 M KCl), collect the eluate (1 ml/fraction; total 8 fractions).
        6. Isocratic flow (0.5 ml/min) with 7.8 ml (6 column volumes) of 100% solvent B (i.e., 0% solvent A; final 1 M KCl), collect the eluate (1 ml/fraction; total 8 fractions).
      4. Make small aliquot(s) of each stepwise-fractionated sample by the UNO-Q column, and freeze in liquid N2 and store at -80 °C.
      5. Check the IREF-2 activity present in each fraction (see Note 10).
    3. Gradient fractionation on a strong anion-exchanger column
      1. Set up the FPLC system and the UNO Q1R column, routinely
        1. Set degassed buffer HG as “solvent A” to inlet A and degassed buffer HGK as “solvent B” to inlet B, respectively.
        2. Wash the column with 100% solvent B, and equilibrate with 25% solvent B (i.e., 75% solvent A).
      2. Thaw the IREF-2-active fractions of the stepwise fractionated sample on the UNO-Q column chromatography (= total 8 ml), the eluate with 0.6 M KCl [i.e., mixture of 60% of buffer HGK (as solvent B) and 40% of buffer HG (as solvent A)], and dilute with 1.5 volume of buffer HG to decrease the salt concentration in the sample (finally, 20 ml as an input volume). Centrifuge at 20,000 x g for 5 min at 4 °C to remove insoluble material.
      3. Load the supernatant into the sample loop and change the valve position to “inject”. Run the program for gradient fractionation on the UNO-Q column (1.3 ml column volume) as follows:
        1. Isocratic flow (0.5 ml/min) with 30 ml (~1.5 volumes of the input) of 25% solvent B (i.e., 75% solvent A; final 0.25 M KCl), collect the eluate (1.5 ml/fraction; total 20 fractions).
        2. Change the valve position from “inject” to “load”.
        3. Linear gradient flow (0.5 ml/min) ranging from 25% to 80% of solvent B with 15 ml, collect the eluate (0.5 ml/fraction; total 30 fractions).
      4. Make small aliquot(s) of each fractionated sample on the UNO-Q column chromatography, and freeze in liquid N2 and store at -80 °C.
      5. Check the IREF-2 activity present in each fraction (see Note 10).
    4. Gradient fractionation on a hydrophobic column chromatography
      1. Prepare the FPLC system and pre-packed hydrophobic column, SOURCE 15PHE PE 4.6/100, routinely
        1. Set degassed buffer HG as solvent A to inlet A and degassed buffer HGN1M as solvent B to inlet B, respectively.
        2. Wash the column with 100% solvent A, and equilibrate with 100% solvent B.
      2. Thaw the IREF-2-active fractions of the gradient-fractionated sample by the UNO-Q column chromatography and combine them as an input sample (= total 3 ml), and mix an equal volume of ice-cold buffer HGN2M, gradually. Centrifuge at 20,000 x g for 5 min at 4 °C to remove insoluble material.
      3. Load the diluted supernatant (~6 ml) into the sample loop and change the valve position to “inject”. Run the program for gradient fractionation on the SOURCE 15PHE column (1.7 ml column volume) by reducing concentration of (NH4)2SO4, as follows: 
        1. Isocratic flow (0.3 ml/min) with 15 ml (~2.5 volumes of the input) of 100% solvent B [i.e., 0% solvent A, final 1 M (NH4)2SO4], collect the eluate (0.75 ml/fraction; total 20 fractions).
        2. Change the valve position from “inject” to “load”.
        3. Linear gradient flow (0.3 ml/min) ranging from 100% to 0% of solvent B (i.e., from 0% to 100% of solvent A) with 12 ml, collect the eluate (0.75 ml/fraction; total 16 fractions).
      4. Dialyze each fraction against 50 volumes of buffer HG for 5 h at 4 °C.
      5. Make small aliquot(s) of each fractionated and dialyzed sample by the hydrophobic column chromatography, and freeze in liquid N2 and store at -80 °C.
      6. Check the IREF-2 activity present in each fraction (see Note 10).
    5. Gradient fractionation on a strong cation-exchanger column chromatography
      1. Set up the FPLC system and pre-packed UNO S1 column, routinely.
        1. Set degassed buffer HG as solvent A to inlet A and degassed buffer HGK as solvent B to inlet B, respectively.
        2. Wash the column with 100% solvent B, and equilibrate with 3% solvent B (i.e., 97% solvent A).
      2. Thaw the IREF-2-active fraction of the hydrophobic column chromatography, (i.e., unbound “flow-through” fraction). Centrifuge at 20,000 x g for 5 min at 4 °C to remove insoluble material.
      3. Load the supernatant (approximately 5 ml) to the sample loop and change the valve position to “inject”. Run the program for gradient fractionation on the UNO-S column (1.3 ml column volume) as follows: 
        1. Isocratic flow (0.4 ml/min) with 10 ml (2 volumes of the input) of 3% solvent B (i.e., 97% solvent A; final 0.03 M KCl), collect the eluate (0.65 ml/fraction; total 16 fractions).
        2. Change the valve position from “inject” to “load”.
        3. Linear gradient flow (0.5 ml/min) ranging from 3% to 100% of solvent B with 15.6 ml (12 column volumes), collect the eluate (0.65 ml/fraction; total 24 fractions).
      4. Make small aliquot(s) of each fractionated sample on the UNO-S column chromatography, and freeze in liquid N2 and store at -80 °C.
      5. Check the IREF-2 activity present in each fraction (see Note 10).
    6. Gradient fractionation on a strong anion-exchanger column
      1. Set up the FPLC system and pre-packed Mono-Q column, routinely.
        1. Set degassed buffer HG as solvent A to inlet A and degassed buffer HGK as solvent B to inlet B, respectively.
        2. Wash the column with 100% solvent B, and equilibrate with 35% solvent B (i.e., 65% solvent A).
      2. Thaw the IREF-2-active fraction from the fractionated samples by the cation exchanger (UNO-S) column chromatography (i.e., unbound “flow-through” fraction). Centrifuge at 20,000 x g for 5 min at 4 °C to remove insoluble material.
      3. Load the supernatant (approximately 5 ml) to the sample loop and change the valve position to “inject”. Run the program for gradient fractionation on the Mono-Q column (1 ml column volume) as follows:
        1. Isocratic flow (0.5 ml/min) with 10 ml (2 volumes of the input) of 35% solvent B (i.e., 65% solvent A; final 0.35 M KCl), collect the eluate (1 ml/fraction; total 10 fractions).
        2. Change the valve position from “inject” to “load”.
        3. Linear gradient flow (0.4 ml/min) ranging from 35% to 70% with 12 ml, collect the eluate (0.4 ml/fraction; total 30 fractions).
      4. Make small aliquot(s) of each fraction from the Mono-Q column chromatography, and freeze in liquid N2 and store at -80 °C.
      5. Check the IREF-2 activity present in each fraction (see Note 10 and Figure 4).


        Figure 4. Profile of the fractions from Mono-Q column chromatography for IREF-2. Each Mono-Q fraction (fraction numbers 1-11) or input material for the Mono-Q column chromatography (i.e., unbound fraction of the UNO-S column chromatography at procedure C, 5) was individually added to the cell-free viral RNA synthesis reaction (upper panel). Each fraction was also subjected to 11.5% SDS-PAGE, and polypeptides were visualized by silver staining (lower panel). The arrow indicates viral RNA replication products. By comparing carefully between the IREF-2 activity level and the elution pattern of each peptide, two peptides (arrowheads A and B) migrated at 31 and 30 kDa are expected to be candidates for the IREF-2 activity. The molecular weight (kDa) positions are denoted on the left side of the panel. MWM: molecular weight maker.

    7. Fractionation on a gel filtration column (see Note 11)
      1. Set up the SMART system and gel filtration column, routinely.
        1. Set degassed buffer HG as solvent A to inlet A and degassed buffer HGK as solvent B to inlet B, respectively.
        2. Wash the column with 100% solvent B, and equilibrate with 20% solvent B (i.e., 80% solvent A).
      2. Thaw the IREF-2-active fraction number 8 of the anion exchanger column chromatography (see Figure 4). Centrifuge at 20,000 x g for 5 min at 4 °C to remove insoluble material.
      3. Load a small part of the supernatant (approximately 40 μl) into the sample loop and change the valve position to “inject”. Run the program for fractionation on the Superdex 75 gel filtration column (2.4 ml column volume) as follows: 
        1. Isocratic flow (50 μl/min) with 2.4 ml (1 column volume) of 20% solvent B (i.e., 80% solvent A; final 0.2 M KCl).
        2. At 0.78 ml of the total flow, start collecting the eluate from the gel filtration column (30 μl/fraction).
      4. Make small aliquot(s) of each fraction on the gel filtration column chromatography, and freeze in liquid N2 and store at -80 °C.
      5. Check the IREF-2 activity present in each fraction (see Note 10 and Figure 5).


        Figure 5. Profile of the fractions from gel filtration chromatography for IREF-2. Each gel-filtrated fraction (fraction numbers 1-23) or input material for the gel-filtration chromatography (i.e., the fraction number 8 of the Mono-Q column chromatography in step C6) was individually added to the cell-free viral RNA synthesis reaction (upper panel). Each fraction was also subjected to 11.5% SDS-PAGE, and polypeptides were visualized by silver staining (lower panel). The arrow indicates viral RNA replication products. The arrowheads indicate two candidate peptides responsible for the IREF-2 activity. The molecular weight (kDa) positions are denoted on the left side of the panel. MWM: molecular weight maker.

  4. Identification of two polypeptides responsible for IREF-2 activity by mass spectrometry
    1. Subject two Mono-Q fractions involving two candidate peptides, the fraction number 6 enriched with the peptide A and the fraction number 9 enriched with the peptide B (see Figure 4), to 11.5% SDS-PAGE.
    2. Visualize the peptides separated in the gel by using a glutaraldehyde-free silver staining kit (for MS analysis).
    3. Excise the gel pieces containing the bands of the peptide A and B, respectively (by using clean razor blade).
    4. Tryptic digestion in the gel pieces, followed by mass analysis (MALDI-TOF MASS).

Data analysis

Finally, pp32 (peptide A) and APRIL (peptide B) were identified as peptides derived from IREF-2 by database search using MS-Fit program in Protein Prospector (University of California San Francisco; http://prospector.ucsf.edu/prospector/mshome.htm). Top 3 hits of each mass analysis were summarized in Table 1. Observed mass (m/z) and theoretical mass for each digested peptide were summarized as “supplementary file 1 (http://elifesciences.org/content/4/e08939v2/supp-material1)” in the original article (Sugiyama et al., 2015).

Table 1. Top 3 hit proteins identified by MALDI-TOF MASS analyses

Notes

  1. Several materials/reagents and equipments listed here, which were actually used in this protocol, are unavailable now. Of course, you can substitute these with other company’s or industry’s products.
  2. For 8-L scale culture, it usually takes ~2 weeks from the beginning.
  3. If more NEs are required for your purpose, a part of the cell culture (e.g., ~200 ml) should be aseptically kept and used for another round of large-scale culture.
  4. All procedures for preparation of the nuclear extracts should be carried out as rapidly as possible at 4 °C or on ice.
  5. Packed cell volume obtained from 8-L scale culture is usually 10-15 ml.
  6. Packed nuclear volume obtained from 8-L scale culture is usually 8-12 ml.
  7. The protein concentration usually results in about 5-10 mg/ml.
  8. It is preferable to proceed continuously to the next procedure (i.e., first fractionation on the phosphocellulose column chromatography in this protocol) without freezing and thawing of NEs. To this end, the phosphocellulose column and FPLC system should be prepared on ahead to be ready-to-use (steps C1a and C1b).
  9. Procedures of column chromatography (e.g., choice of column and fractionation way in stepwise or gradient, etc.) must be highly dependent on the chromatographic behavior and the purity of the protein of your interest at each column chromatography step. In addition to the column chromatography, you can utilize “ammonium sulfate precipitation” or “(glycerol or sucrose) density gradient centrifugation” method. For other cases of purification procedures, refer also to previous reports from our laboratory (Momose et al., 2001; Okuwaki et al., 2001; Momose et al., 2002; Kawaguchi and Nagata, 2007) and other laboratories.
  10. Cell-free influenza virus RNA replication assay is carried out, as described briefly below: Single reaction (final volume of 20 μl) contains 50 mM HEPES-NaOH (pH 7.9), 3 mM MgCl2, 50 mM KCl, 1 mM DTT, 500 μM each ATP, UTP and CTP and 25 μM GTP, 5 mCi of [α-32P] GTP (3,000 Ci/mmol), 8 U of RNase inhibitor from human placenta, 10 ng of 53 nt-long influenza virus artificial model RNA template (termed “cRNA”) and 58 fmol of the viral RNA-dependent RNA polymerase prepared from influenza virion particles (termed “mnRNP”) as an enzyme source. In addition, NE fractions at each column chromatography step were individually added in the reactions to address whether IREF-2 activity was involved in the fraction, or not. After incubation at 30 °C for 2 h, reactions were terminated by extraction with phenol/chloroform followed by precipitation of RNA products with ethanol. The precipitated materials were subjected to 10% polyacrylamide gel electrophoresis in the presence of 7 M urea (Urea-PAGE), and visualized by autoradiography. For the detail of the cell-free viral RNA replication assay, see Sugiyama et al. (2015).
  11. This gel filtration chromatography was performed for further confirmation that the peptides A and B (see Figure 4) were really responsible for the IREF-2 activity, not for further purification. Therefore, a small part of the IREF-2 active fraction (fraction number 8 in Figure 4) was subjected to the gel filtration chromatography with the Micro-Purification SMART system.

Recipes

Note: All buffers listed below should be prepared before use (not prepared before long time) as ultra-pure “nuclease-free” grade.

  1. Buffer A (used in steps B3 and B5)
    10 mM HEPES-NaOH (pH 7.9 at 4 °C)
    10 mM KCl
    1.5 mM MgCl2
    0.5 mM DTT (add 1 M stock just before use)
  2. Buffer C (used in step B10)
    20 mM HEPES-NaOH (pH 7.9 at 4 °C)
    420 mM NaCl
    1.5 mM MgCl2
    200 mM EDTA
    25% (v/v) glycerol
    0.5 mM PMSF [add 1 M stock (in DMSO) just before use]
    0.5 mM DTT (add 1 M stock just before use)
  3. Buffer D (used in step B14)
    20 mM HEPES-NaOH (pH 7.9 at 4 °C)
    100 mM KCl
    12.5 mM MgCl2
    200 mM EDTA
    25% (v/v) glycerol
    0.5 mM PMSF [add 1M stock (in DMSO) just before use]
    0.5 mM DTT (add 1M stock just before use)
  4. Buffer HG (used in step C)
    50 mM HEPES-NaOH (pH 7.9 at 4 °C)
    20% (v/v) glycerol
    1. Filtrate and degas with bottle top filter under vacuum.
    2. Chill in 4 °C.
    3. Add DTT (1 M stock, final 1 mM) just before use.
  5. Buffer HGK (used in step C)
    50 mM HEPES-NaOH (pH 7.9 at 4 °C)
    1 M KCl
    20% (v/v) glycerol
    1. Filtrate and degas with bottle top filter under vacuum.
    2. Chill in 4 °C.
    3. Add DTT (1 M stock, final 1 mM) just before use.
  6. Buffer HGN1M (used in step C4)
    50 mM HEPES-NaOH (pH 7.9 at 4 °C)
    1 M (NH4)2SO4
    20% (v/v) glycerol
    1. Filtrate and degas with bottle top filter under vacuum.
    2. Chill in 4 °C.
    3. Add DTT (1 M stock, final 1 mM) just before use.
  7. Buffer HGN2M (used in step C4b)
    50 mM HEPES-NaOH (pH 7.9 at 4 °C)
    2 M (NH4)2SO4
    20% (v/v) glycerol
    1 mM DTT (add 1 M stock just before use)

Acknowledgments

The protocol for preparation of NEs preparation was based on Dignam et al. (1983). This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to K. N.).

References

  1. Dignam, J. D., Lebovitz, R. M. and Roeder, R. G. (1983). Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11(5): 1475-1489.
  2. Kawaguchi, A. and Nagata, K. (2007). De novo replication of the influenza virus RNA genome is regulated by DNA replicative helicase, MCM. EMBO J 26(21): 4566-4575.
  3. Matsumoto, K., Nagata, K., Ui, M. and Hanaoka, F. (1993). Template activating factor I, a novel host factor required to stimulate the adenovirus core DNA replication. J Biol Chem 268(14): 10582-10587.
  4. Momose, F., Basler, C. F., O'Neill, R. E., Iwamatsu, A., Palese, P. and Nagata, K. (2001). Cellular splicing factor RAF-2p48/NPI-5/BAT1/UAP56 interacts with the influenza virus nucleoprotein and enhances viral RNA synthesis. J Virol 75(4): 1899-1908.
  5. Momose, F., Naito, T., Yano, K., Sugimoto, S., Morikawa, Y. and Nagata, K. (2002). Identification of Hsp90 as a stimulatory host factor involved in influenza virus RNA synthesis. J Biol Chem 277(47): 45306-45314.
  6. Nagata, K., Kawaguchi, A. and Naito, T. (2008). Host factors for replication and transcription of the influenza virus genome. Rev Med Virol 18(4): 247-260.
  7. Okuwaki, M., Iwamatsu, A., Tsujimoto, M. and Nagata, K. (2001). Identification of nucleophosmin/B23, an acidic nucleolar protein, as a stimulatory factor for in vitro replication of adenovirus DNA complexed with viral basic core proteins. J Mol Biol 311(1): 41-55.
  8. Simon, R. H. and Felsenfeld, G. (1979). A new procedure for purifying histone pairs H2A + H2B and H3 + H4 from chromatin using hydroxylapatite. Nucleic Acids Res 6(2): 689-696.
  9. Schnitzler, G. R. (2001). Isolation of histones and nucleosome cores from mammalian cells. Curr Protoc Mol Biol Chapter 21: Unit 21 25.
  10. Sugiyama, K., Kawaguchi, A., Okuwaki, M. and Nagata, K. (2015). pp32 and APRIL are host cell-derived regulators of influenza virus RNA synthesis from cRNA. Elife 4: e08939.

材料和试剂

  1. 组织培养皿
  2. 离心管[例如,对于F0650转子(Beckman Coulter,目录号:363075)]
  3. 锥形离心管(聚丙烯,50ml)(例如,Corning,目录号:430829)
  4. 锥形离心管(聚丙烯,15ml)(例如,Corning,目录号:430791)
  5. 透析膜管(纤维素膜,14,000道尔顿分子量截留),(EIDIA,目录号:UC27-32-100)
  6. 瓶顶过滤器(Merck Millipore Corporation,目录号:SCGPS01RE)
  7. 微离心管(聚丙烯,1.5ml大小和0.6ml大小)(例如,Eppendorf)
  8. HeLa S3细胞[National Institutes of Biomedical Innovation,Japanese Collection of Research Bioresources(JCRB),目录号:JCRB0713]
  9. 最小必需培养基Eagle(MEM)(Sigma-Aldrich,目录号:M4655)
  10. 将致死性牛血清(FBS)(例如Thermo Fisher Scientific,Gibco TM ,目录号:26140-079)
  11. S-MEM [Sigma-Aldrich,目录号:M4767(停产,见注1)。 或者,也可参见Sigma-Aldrich,目录号:M8167,或其他公司的类似产品]
  12. 牛血清(CS)(例如Thermo Fisher Scientific,Gibco TM ,目录号:16170-078)
  13. 超纯水(Milli-Q)
  14. 磷酸盐缓冲盐水(PBS)
  15. 2- [4-(2-羟乙基)-1-哌嗪基]乙磺酸(HEPES)缓冲液
  16. 氢氧化钠(NaOH)
  17. 盐酸(HCl)
  18. 氯化钠(NaCl)
  19. 氯化钾(KCl)
  20. 氯化镁六水合物(MgCl 2)
  21. 二硫苏糖醇(DTT)
  22. 胰蛋白酶-EDTA(用于细胞胰蛋白酶消化)
  23. 甘油
  24. 二钠二氢乙二胺四乙酸二水合物(EDTA)
  25. 苯甲基磺酰氟(PMSF)
  26. 二甲基亚砜(DMSO)
  27. 液体N <2>
  28. Whatman阳离子交换纤维素(P11)(Sigma-Aldrich,目录号:4071010)
  29. 硫酸铵[(NH 4)2 SO 4 4]
  30. 银染试剂盒(例如,Cosmo Bio,目录号:DCB-423413)
  31. 银染试剂盒(无甲醛的MS分析)(例如,Wako Pure Chemical industries,目录号:299-58901)
  32. 缓冲区A(参见配方)
  33. 缓冲区C(参见配方)
  34. 缓冲区D(参见配方)
  35. 缓冲液HG(参见配方)
  36. 缓冲区HGK(参见配方)
  37. 缓冲区HGN 1M (请参阅配方)
  38. 缓冲区HGN 2M (请参阅配方)

设备

  1. 细胞培养环境和设备
    1. 清洁长椅
    2. CO 2培养箱(37℃,用于附着培养)
    3. 显微镜(相位对比)
    4. 血细胞计数器
    5. 旋转瓶(小尺寸〜500ml,大尺寸8L)
    6. 磁力搅拌器(速度慢;旋转瓶尺寸足够)
    7. 37℃培养箱(足够尺寸的旋转瓶和磁力搅拌器)
    8. 高压灭菌器(旋转瓶尺寸足够)
  2. 大规模离心机(例如,Beckman Coulter,型号:Avanti J-26XP)
  3. 大规模离心转子(Beckman Coulter,型号:JLA-10.500)
  4. 离心瓶(500ml大小,用于JLA-10.500转子)(例如,Beckman Coulter,目录号:361691)
  5. 台式高速离心机(例如,Beckman Coulter,型号:Allegra 64R)
  6. 高速离心机转子[例如,F0650(Beckman Coulter,目录号:364610)]
  7. 草ce匀浆器,15ml(Capitol Scientific,Wheaton ,目录号:357544)
  8. 草ce匀浆器用杵,40ml(Capitol Scientific,Wheaton ,目录号:357546)
  9. 冰箱(透析)
  10. 深冷冻(-80℃)
  11. pH计
  12. 草瓶(Pyrex ,干热可灭菌)
  13. 台式离心机
  14. SDS-PAGE装置
  15. 柱色谱设备(见注1)
    1. 快速蛋白质液相色谱(FPLC)工作站(Bio-Rad Laboratories,型号:BioLogic HR)
    2. 色谱室(4℃)
    3. 柱外壳(例如,Bio-Rad Laboratories,Econo Column ,目录号:7371512)
    4. 流量适配器(例如,Bio-Rad Laboratories,目录号:7380016)
    5. 阴离子交换剂,UNO TM Q1R(Bio-Rad Laboratories,目录号:7200011)
    6. 疏水柱,SOURCE 15PHE PE 4.6/100(GE Healthcare,目录号:17-5186-01)
    7. 阳离子交换剂,UNO TM S1(Bio-Rad Laboratories,目录号:7200021)
    8. 阴离子交换剂,Mono Q HR5/5 [GE Healthcare,型号:Mono Q HR 5/5(停止生产)。或者,可以使用Mono Q 5/50GL(GE Healthcare,型号:17-5166-01)或来自其他公司的类似产品]
    9. 微量净化SMART系统[GE Healthcare,Pharmacia Biotech,型号:SMART 系统(生产停止)]
    10. 凝胶过滤柱(GE Healthcare,Pharmacia Biotech,型号:Superdex 75 PC 3.2/30)
      注意:此产品已停产。
  16. 剃刀刀片

程序

  1. HeLa S3细胞的大规模培养(见注2)
    1. 种子HeLa S3细胞在组织培养皿(es) 在MEM + 10%FBS培养基中。
    2. 在37℃下在5%CO 2中孵育以生长,直到总细胞数达到≥1×10 7个为止。保持粘附细胞的密度在30-80%汇合。如果必要,通过增加培养皿的大小和/或数量来传代所有生长的细胞。
    3. 胰蛋白酶消化细胞,重悬于S-MEM + 5%小牛血清(CS)中,并计数细胞数。
    4. 通过加入S-MEM + 5%CS将细胞密度调节至约2×10 5个细胞/ml。
    5. 将细胞悬浮液放入500ml大小的旋转瓶(灭菌)中,并在37℃下通过磁力搅拌器(60-80转/分钟)温和搅拌孵育。
    6. 通过每日计数细胞数目来监测细胞生长,并加入新鲜培养基(S-MEM + 5%CS)以维持细胞在3-6×10 5个细胞/ml。当细胞培养物的体积达到500ml时,将培养物转移到大规模旋转瓶(8L大小)中并在37℃下保持培养。
    7. 当细胞培养物达到最大体积(例如,8μL的6×10 5个细胞/ml;大约5×10 9个总细胞/ml)时,细胞),进行核提取物制备步骤(参见注释3)
  2. 从HeLa S3细胞制备核提取物(NE)(参见注释4)
    1. 通过离心(1500×g,4℃,10分钟)收获培养的HeLa S3细胞。
    2. 将细胞沉淀悬浮在PBS中,并通过离心收集
    3. 将细胞悬浮在冰冷的缓冲液A的约5个填充细胞体积(PCV)(即,所收集细胞的团块质量)中,并在冰上储存10分钟(参见注释5)。
    4. 通过在4℃下以1500×g离心10分钟收集
    5. 将细胞悬浮在缓冲液A的2个PCV中,使细胞在冰上膨胀10分钟
    6. 用"B型(紧密)"杵通过10-15冲程的dounce匀浆器裂解细胞
    7. 检查细胞裂解在显微镜下(图1)。如果均质化不足,再次均质化并再次检查裂解

      图1.细胞匀浆的微观视图。通过相差显微镜在200倍放大下观察均质化之前和之后的悬浮液(步骤B6)。 A.悬浮在PBS中的细胞(步骤B2); B.在匀浆化之前在低渗"缓冲液A"中使肿胀细胞(步骤B5); C.匀浆的细胞悬浮液(步骤B6)。观察到细胞质碎片和细胞核
    8. 在4℃下以3,300×g离心匀浆10分钟,除去上清液。在该步骤除去的上清液可以保存为"S-100"细胞质提取物(Dignam等人,1983),并用于其它目的(Matsumoto等人)。 ,1993)。
    9. 在4℃下以14,000×g离心离心剩余的沉淀(即,核),持续20分钟,并完全除去残余的上清液。
    10. 用冰冷的缓冲液C(参见配方和注释6)将细胞悬浮在等体积的填充细胞核中(即等于所收集的细胞核的沉淀质量),并通过10次冲击均质机与"B型(紧)"杵,以确保完全悬浮
    11. 将匀浆转移到离心管中,通过在4℃下旋转30分钟轻轻地倒转
    12. 在4℃下以25,000×g离心匀浆10分钟
    13. 将上清液(NEs)放置在透析膜袋中。在此步骤中剩余的沉淀可以冷冻并储存在-80℃下,并用作染色质级分(Simon和Felsenfeld,1979; Schnitzler,2001)。
    14. 在4℃下用≥50体积的缓冲液D透析3小时。将缓冲液更换为新鲜缓冲液D(≥50体积),另外在4℃下透析3小时
    15. 将透析液(白色NEs)转移到离心管中,并通过台式离心机以25,000×g离心20分钟。
    16. 确定上清液NEs的蛋白质浓度(见注7)。将分配等分试样分配到管中(如果需要),并在液体N 2中冷冻并储存在-80℃直到使用(参见注释8)。

  3. 通过连续柱层析分离和纯化NE(参见图2和注释9)。本文中,需要柱色谱法的程序来纯化和鉴定称为"流感病毒复制因子(IREF)-2"的两种宿主衍生的因子pp32和APRIL(分别称为ANP32A和B),其刺激流感病毒RNA复制过程(Sugiyama等,2015)。


    图2. NEEF的IREF-2的纯化方案。IREF-2的色谱行为似乎是高度酸性和亲水性的。

    1. 弱阳离子交换柱上的逐步分馏
      1. 准备磷酸纤维素(PC)柱,如下:
        1. 通过在≥20体积的水中轻轻旋转并保持30〜60分钟来悬浮PC树脂的粉末(Whatman 阳离子交换纤维素P11)。
        2. 倒出上清液,将树脂重悬于水中,并重复至少8-10次以除去任何未沉淀的细粒。
        3. 将树脂悬浮在0.2M NaOH中,静置30分钟
        4. 倒出上清液并重复,直至上清液的pH高于10
        5. 用水洗涤树脂,直到上清液的pH值低于8
        6. 将树脂悬浮在0.2M HCl中,使其沉降
        7. 倒出上清液并重复,直到上清液的pH低于3
        8. 用水洗涤树脂,直到上清液的pH值约为5
        9. 平衡缓冲液HG中的树脂。 如果PC树脂存储超过一周,加入0.1%叠氮化钠并储存在4°C(从不冷冻)。
        10. 悬浮PC树脂,并小心地倒入柱外壳[约10毫升(床体积)的PC树脂足够10〜15毫升的输入NEs],并允许沉降。 然后,将流量适配器小心地安装在床上。
      2. 设置FPLC系统和柱,常规如下:
        1. 用纯脱气水清洗系统(包括"样品回路")。
        2. 将准备的PC柱(10ml床体积)连接到系统,不引入空气,并用足够的水清洗系统(包括色谱柱)。 确保在流动过程中没有柱的渗漏。
        3. 将脱气缓冲液HG设为溶剂A至入口A,将脱气缓冲液HGK设为溶剂B至入口B.
        4. 用100%溶剂B(缓冲液HGK; 1M KCl)洗涤系统,然后用5%溶剂B(即95%缓冲液HG的溶剂A; 50mM KCl)平衡。
      3. 确保阀门位置为"负载",并将NE(在本协议中约12.5 ml)装入样品回路。
      4. 将阀门位置更改为"注射",然后运行程序以在PC柱(10ml床体积)上开始逐步分馏,如下所示:
        1. 用45ml(4体积的输入NE)5%溶剂B(即95%溶剂A;最终0.05M KCl)的等度流动(0.15ml/min)收集洗脱液2ml /级分;共23份)
        2. 将阀门位置从"进样"更改为"加载",以绕过样品回路的流路。如果忘记了,以下盐洗脱步骤将延迟样品环的体积。
        3. 用40ml(4柱体积)的20%溶剂B(即80%溶剂A;最终0.2M KCl)的等度流动(0.15ml/min)收集洗脱液(2ml /分数;总共20个分数)
        4. 用40ml(4柱体积)50%溶剂B(即50%溶剂A;最终0.5M KCl)的等度流速(0.15ml/min)收集洗脱液(2ml /分数;总共20个分数)
        5. 用40ml(4柱体积)的100%溶剂B(即0%溶剂A;最终1M KCl)的等度流动(0.15ml/min)收集洗脱液(2ml /部分;总共20个部分)。
      5. 使每个分级样品的小等分试样避免将来不必要的冻融循环,并在液氮中冻结并储存在-80℃。
      6. 通过使用无细胞的病毒RNA复制测定,检查参与PC级分的活性(,即流感病毒RNA复制的刺激活性,在本方案中称为"IREF-2"),小等分(见图3和注10)

        图3.来自IREF-2的磷酸纤维素柱层析的级分概况将来自磷酸纤维素(PC)柱层析的NE的每个逐步级分分别添加到无细胞病毒RNA复制反应中(1.75 μg各级分/反应),如下: (泳道1)洗脱的未结合部分(流出物),用含有0.2M KCl(泳道2),0.5M KCl(泳道3)和1μMKCl的泳道洗脱的材料M KCl(泳道4)。关于无细胞病毒RNA复制测定的细节,参见注释10和原始文章(Sugiyama等人,2015)。检测到鲁棒的病毒RNA产物(上图,泳道1),表明刺激病毒RNA复制反应的某些活性,称为IREF-2,存在于来自PC柱层析的流过级分中。每个级分还进行14%SDS-PAGE(每个级分/泳道1μg),并通过银染色显示多肽(下图)。箭头表示病毒RNA复制产物。分子量(kDa)位置表示在下图的右侧。 MWM:分子量制造商
    2. 在强阴离子交换柱上进行分步分馏
      1. 定期设置FPLC系统和预先包装的UNO Q1R柱
        1. 将脱气缓冲液HG设为溶剂A至入口A,将脱气缓冲液HGK设为溶剂B至入口B.
        2. 用100%溶剂B洗涤柱,并用5%溶剂B(即95%溶剂A)平衡。
      2. 解冻PC柱层析(即未结合的"流通"级分,参见图3)的IREF-2活性阳性级分(本方案中总共9ml),并在20,000 xg 在4°C下5分钟,以去除不溶物
      3. 将上清液装入样品环,并将阀位置更改为"注射"。运行程序以在UNO-Q柱(1.3ml柱体积)上开始逐步分级,如下:
        1. 使用18ml(2体积输入)5%溶剂B(即95%溶剂A;最终0.05M KCl)的等度流动(0.5ml/min)收集洗脱液ml /分数;总共18个分数)
        2. 将阀门位置从"进样"更改为"加载"
        3. 用7.8ml(6柱体积)15%溶剂B(即85%溶剂A;最终0.15M KCl)的等度流速(0.5ml/min)收集洗脱液(1ml /分数;总共8个分数)
        4. 用7.8ml(6柱体积)的30%溶剂B(即70%溶剂A;最终0.3M KCl)的等度流速(0.5ml/min)收集洗脱液(1ml /分数;总共8个分数)
        5. 用7.8ml(6柱体积)的60%溶剂B(即40%溶剂A;最终0.6M KCl)的等度流动(0.5ml/min)收集洗脱液(1ml /分数;总共8个分数)
        6. 使用18ml(2体积输入)5%溶剂B(即95%溶剂A;最终0.05M KCl)的等度流动(0.5ml/min)收集洗脱液ml /分数;总共18个分数)
        7. 将阀门位置从"进样"更改为"加载"
        8. 用7.8ml(6柱体积)15%溶剂B(即85%溶剂A;最终0.15M KCl)的等度流速(0.5ml/min)收集洗脱液(1ml /分数;总共8个分数)
        9. 用7.8ml(6柱体积)的30%溶剂B(即70%溶剂A;最终0.3M KCl)的等度流速(0.5ml/min)收集洗脱液(1ml /分数;总共8个分数)
        10. 用7.8ml(6柱体积)的60%溶剂B(即40%溶剂A;最终0.6M KCl)的等度流动(0.5ml/min)收集洗脱液(1ml /分数;总共8个分数)
        11. ...
        12. 定期设置FPLC系统和UNO Q1R列
          1. 将脱气缓冲液HG设定为"溶剂A"至入口A,将脱气缓冲液HGK设定为"溶剂B"至入口B。
          2. 用100%溶剂B洗涤柱,并用25%溶剂B(即75%溶剂A)平衡。
        13. 在UNO-Q柱色谱(=总共8ml)上将分步分馏的样品的IREF-2活性级分解冻,将具有0.6M KCl的洗脱液[即60%缓冲液HGK(作为溶剂B)和40%的缓冲液HG(作为溶剂A)],并用1.5体积的缓冲液HG稀释以降低样品中的盐浓度(最后,作为输入体积为20ml)。在4℃下以20,000×g离心5分钟以除去不溶性物质。
        14. 将上清液装入样品环,并将阀位置更改为"注射"。在UNO-Q柱(1.3ml柱体积)上进行梯度分馏的程序如下:
          1. 用30ml(〜1.5体积的输入)25%溶剂B(即75%溶剂A;最终0.25M KCl)的等度流动(0.5ml/min)收集洗脱液1.5ml /级分;共20份)
          2. 将阀门位置从"进样"更改为"加载"
          3. 从25%至80%的溶剂B与15ml的线性梯度流(0.5ml/min)收集洗脱液(0.5ml /级分;总共30个级分)。
        15. 在UNO-Q柱色谱上制备每个分级样品的小等分试样,并在液氮中冷冻并保存在-80℃。
        16. 检查每个级分中存在的IREF-2活性(参见注释10)。
      4. 在疏水柱色谱上的梯度分馏
        1. 准备FPLC系统和预填充疏水柱,SOURCE 15PHE PE 4.6/100,常规
          1. 设置脱气缓冲液 HG作为溶剂A至入口A,并将脱气缓冲液HGN 1M作为溶剂B至入口B.
          2. 用100%溶剂A洗涤柱,并用100%溶剂B平衡。
        2. 通过UNO-Q柱层析解冻梯度分级的样品的IREF-2活性级分,并将其作为输入样品(=总共3ml)合并,并混合等体积的冰冷缓冲液HGN 。在4℃下以20,000×g离心5分钟以除去不溶性物质
        3. 将稀释的上清液(〜6ml)装入样品环,并将阀位置改变为"注射"。运行程序,通过降低(NH 4)2 SO 4的浓度,在SOURCE 15PHE柱(1.7ml柱体积)上进行梯度分级,如下所示:
          1. 用15ml(〜2.5体积的输入量)100%溶剂B [即0%溶剂A,最后1M(NH 4)]的等度流速(0.3ml /收集洗脱液(0.75ml /级分;总共20个级分)。
          2. 将阀门位置从"进样"更改为"加载"
          3. 用12ml从100%至0%溶剂B(即从0%至100%的溶剂A)的线性梯度流(0.3ml/min)收集洗脱液(0.75ml /部分;总共16个部分)。
        4. 在4℃下用50体积的缓冲液HG透析每个级分5小时
        5. 使每个的小等分试样 通过疏水柱层析分离和透析样品,并在液氮中冷冻并储存在-80℃。
        6. 检查每个级分中存在的IREF-2活性(参见注释10)。
      5. 在强阳离子交换柱色谱上的梯度分级
        1. 定期设置FPLC系统和预先包装的UNO S1色谱柱。
          1. 将脱气缓冲液HG设为溶剂A至入口A,将脱气缓冲液HGK设为溶剂B至入口B.
          2. 用100%溶剂B洗涤柱,并用3%溶剂B(即97%溶剂A)平衡。
        2. 解冻疏水柱层析的IREF-2活性级分(即未结合的"流通"级分)。在4℃下以20,000×g离心5分钟以除去不溶性物质
        3. 加载上清液(约5毫升)到样品环,并改变阀位置为"注射"。在UNO-S柱(1.3 ml柱体积)上运行梯度分馏程序,如下所示:
          1. 使用10ml(2体积输入)的3%溶剂B(即,97%溶剂A;最终0.03M KCl)的等度流动(0.4ml/min)收集洗脱液ml /分数;总共16个分数)
          2. 将阀门位置从"进样"更改为"加载"
          3. 使用15.6ml(12个柱体积)的3%至100%溶剂B的线性梯度流(0.5ml/min)收集洗脱液(0.65ml /级分;总共24个级分)。
        4. 在UNO-S柱色谱上制备每个分级样品的小等分试样,并在液氮中冷冻并储存在-80℃。
        5. 检查每个级分中存在的IREF-2活性(参见注释10)。
      6. 在强阴离子交换柱上的梯度分馏
        1. 定期设置FPLC系统和预包装的Mono-Q柱。
          1. 将脱气缓冲液HG设为溶剂A至入口A,将脱气缓冲液HGK设为溶剂B至入口B.
          2. 用100%溶剂B洗涤柱,并用35%溶剂B(即65%溶剂A)平衡。
        2. 通过阳离子交换剂(UNO-S)柱色谱(即未结合的"流通"级分)从分级样品中解冻IREF-2-活性级分。在4℃下以20,000×g离心5分钟以除去不溶性物质
        3. 加载上清液(约5毫升)到样品环,并改变阀位置为"注射"。运行程序在Mono-Q柱(1ml柱体积)上梯度分级,如下:
          1. 使用10ml(2体积输入)35%溶剂B(即65%溶剂A;最终0.35M KCl)的等度流动(0.5ml/min)收集洗脱液(1 ml /级分;共10个级分)
          2. 将阀门位置从"进样"更改为"加载"
          3. 用12ml的线性梯度流(0.4ml/min)从35%至70%,收集洗脱液(0.4ml /级分;总共30个级分)。
        4. 使来自Mono-Q柱层析的每个级分的小等分试样,并在液氮中冷冻并储存在-80℃。
        5. 检查每个级分中存在的IREF-2活性(参见注释10和图4)

          图4.来自IREF-2的Mono-Q柱层析的级分的轮廓。每个Mono-Q级分(级分数目1-11)或Mono-Q柱层析的输入材料(
      7. 凝胶过滤柱上的分馏(参见注释11)
        1. 定期设置SMART系统和凝胶过滤柱。
          1. 将脱气缓冲液HG设为溶剂A至入口A,将脱气缓冲液HGK设为溶剂B至入口B.
          2. 用100%溶剂B洗涤柱,并用20%溶剂B(即80%溶剂A)平衡。
        2. 解冻阴离子交换柱层析的IREF-2活性级分8(见图4)。在4℃下以20,000×g离心5分钟以除去不溶性物质
        3. 加载一小部分的上清液(大约40微升)到样品环,并改变阀位置为"注射"。在Superdex 75凝胶过滤柱(2.4 ml柱体积)上运行程序进行分馏,如下:
          1. 用2.4ml(1柱体积)的20%溶剂B(即80%溶剂A;最终0.2M KCl)的等度流动(50μl/min)。
          2. 在0.78ml总流量下,开始收集来自凝胶过滤柱的洗脱液(30μl/级分)。
        4. 在凝胶过滤柱层析上使每个级分的小等分试样,并在液氮中冷冻并储存在-80℃。
        5. 检查每个级分中存在的IREF-2活性(参见注释10和图5)

          图5. IREF-2的凝胶过滤色谱的级分图 每个凝胶过滤级分(级分数1-23)或单独地将用于凝胶过滤层析的输入材料(步骤C6中的Mono-Q柱层析的级分数8)添加到无细胞病毒RNA合成反应(上图) 。每个级分也进行11.5%SDS-PAGE,并且通过银染色显现多肽(下图)。箭头表示病毒RNA复制产物。箭头表示负责IREF-2活性的两种候选肽。分子量(kDa)位置表示在面板的左侧。 MWM:分子量制造商
    3. 鉴定负责IREF-2的两种多肽 质谱的活性
      1. 主题两个涉及两个候选肽的Mono-Q级分,富集肽A的级分6和富集肽B的级分9(见图4)到11.5%SDS-PAGE。
      2. 通过使用无戊二醛银染色试剂盒(用于MS分析)可视化凝胶中分离的肽
      3. 分别切除含有肽A和B的条带的凝胶片(通过使用干净的剃刀刀片)。
      4. 在凝胶块中进行胰蛋白酶消化,然后进行质量分析(MALDI-TOF MASS)

    数据分析

    最后,使用Protein Prospector(University of California San Francisco; http://prospector.ucsf.edu/prospector/mshome.htm )。每个质量分析的前3个命中汇总在表1中。每个消化肽的观察质量(m/z)和理论质量总结为"补充文件1( http://elifesciences.org/content/4/e08939v2/supp-material1 ) ",原文(Sugiyama等人,2015年)。

    表1.通过MALDI-TOF MASS分析确定的前3个命中蛋白

    笔记

    1. 这里列出的几种材料/试剂和设备,实际上在这个协议中使用,现在不可用。 当然,您可以用其他公司或行业的产品代替这些
    2. 对于8-L规模的培养,通常需要大约2个星期从开始
    3. 如果为了您的目的需要更多的NE,则应当将一部分细胞培养物(例如,〜200ml)无菌地保存并用于另一轮大规模培养。
    4. 所有核提取物的制备程序应尽可能在4℃或冰上进行
    5. 从8-L规模培养物获得的包装细胞体积通常为10-15ml
    6. 从8升规模培养物获得的包装核体积通常为8-12ml
    7. 蛋白质浓度通常约为5-10mg/ml
    8. 优选连续进行下一步骤(即,在该方案中在磷酸纤维素柱层析上的第一次分级),而不冻结NEs。为此,应当准备好磷酸纤维素柱和FPLC系统以备即用(步骤C1a和C1b)。
    9. 柱色谱法的步骤(例如,步骤或梯度中的柱选择和分级方法,等)必须高度依赖于色谱行为和蛋白质的纯度您对每个柱色谱步骤感兴趣。除了柱色谱,您可以使用"硫酸铵沉淀"或"(甘油或蔗糖)密度梯度离心"方法。对于其它纯化程序的情况,还参考来自我们实验室的先前报告(Momose等人,2001; Okuwaki等人,2001; Momose等人。,2002; Kawaguchi and Nagata,2007)和其他实验室
    10. 如下简要所述进行无细胞流感病毒RNA复制测定:单一反应(20μl的终体积)含有50mM HEPES-NaOH(pH 7.9),3mM MgCl 2,50 1mM DTT,500μM的各种ATP,UTP和CTP和25μMGTP,5mCi的[α-32 P] GTP(3,000Ci/mmol),8U的RNase抑制剂人类胎盘,10ng的53nt-流感病毒人工模型RNA模板(称为"cRNA")和58fmol从流感病毒颗粒(称为"mnRNP")制备的病毒RNA依赖性RNA聚合酶作为酶源。此外,在每个柱层析步骤中的NE级分单独添加在反应中以说明IREF-2活性是否参与级分。在30℃温育2小时后,通过用苯酚/氯仿萃取终止反应,随后用乙醇沉淀RNA产物。将沉淀的材料在7M尿素(尿素-PAGE)存在下进行10%聚丙烯酰胺凝胶电泳,并通过放射自显影进行显影。对于无细胞病毒RNA复制测定的细节,参见Sugiyama等人。 (2015)。
    11. 进行该凝胶过滤层析以进一步证实肽A和B(参见图4)真正负责IREF-2活性,而不是进一步纯化。因此,将一小部分IREF-2活性级分(图4中的级分数8)进行使用Micro-Purification SMART系统的凝胶过滤色谱法。

    食谱

    注意:以下列出的所有缓冲液应在使用前(未长时间制备)作为超纯"无核酸酶"级别制备。

    1. 缓冲区A(用于步骤B3和B5)
      10mM HEPES-NaOH(pH7.9,4℃) 10 mM KCl
      1.5mM MgCl 2·h/v 0.5mM DTT(在使用前加入1M储备液)
    2. 缓冲区C(在步骤B10中使用)
      20mM HEPES-NaOH(pH7.9,4℃) 420 mM NaCl 1.5mM MgCl 2·h/v 200 mM EDTA
      25%(v/v)甘油 0.5mM PMSF [在使用前加入1M储备液(在DMSO中)]
      0.5mM DTT(在使用前加入1M储备液)
    3. 缓冲区D(在步骤B14中使用)
      20mM HEPES-NaOH(pH7.9,4℃) 100 mM KCl
      12.5mM MgCl 2·h/v 200 mM EDTA
      25%(v/v)甘油 0.5mM PMSF [在使用前加入1M储备液(在DMSO中)]
      0.5mM DTT(在使用前加入1M储备液)
    4. 缓冲液HG(用于步骤C)
      50mM HEPES-NaOH(pH7.9,4℃) 20%(v/v)甘油
      1. 在真空下用瓶顶过滤器过滤和脱气。
      2. 在4°C冷却。
      3. 在使用前加入DTT(1M储备液,最终1mM)。
    5. 缓冲液HGK(用于步骤C)
      50mM HEPES-NaOH(pH7.9,4℃) 1 M KCl
      20%(v/v)甘油
      1. 滤液和脱气 瓶顶过滤器在真空下
      2. 在4°C冷却。
      3. 在使用前加入DTT(1M储备液,最终1mM)。
    6. 缓冲区HGN 1M (在步骤C4中使用)
      50mM HEPES-NaOH(pH7.9,4℃) 1 M(NH 4)2 SO 2 4
      20%(v/v)甘油
      1. 在真空下用瓶顶过滤器过滤和除气
      2. 在4°C冷却。
      3. 在使用前加入DTT(1M储备液,最终1mM)。
    7. 缓冲区HGN <2m>(在步骤C4b中使用)
      50mM HEPES-NaOH(pH7.9,4℃) 2 M(NH 4)2 SO 4 4
      20%(v/v)甘油 1mM DTT(在使用前加入1M储备液)

    致谢

    制备NEs的方案基于Dignam等人。 (1983)。 这项工作部分得到了日本教育,文化,体育,科学和技术部(向K.N.)的赠款援助。

    参考文献

    1. Dignam,JD,Lebovitz,RMand Roeder,RG(1983)。  通过RNA聚合酶II在来自分离的哺乳动物核的可溶性提取物中的精确转录起始。核酸研究 11(5):1475-1489。
    2. Kawaguchi,A.and Nagata,K.(2007)。 
    3. Matsumoto,K.,Nagata,K.,Ui,M.and Hanaoka,F。(1993)。  模板激活因子I,刺激腺病毒核心DNA复制所需的新的宿主因子。 J Biol Chem 268(14):10582- 10587.
    4. Momose,F.,Basler,CF,O'Neill,RE,Iwamatsu,A.,Palese,P。和Nagata,K。(2001)。  细胞剪接因子RAF-2p48/NPI-5/BAT1/UAP56与流感病毒核蛋白相互作用并增强病毒RNA合成。 > 75(4):1899-1908。
    5. Momose,F.,Naito,T.,Yano,K.,Sugimoto,S.,Morikawa,Y.and Nagata,K.(2002)。  Hsp90作为参与流感病毒RNA合成的刺激性宿主因子的鉴定。 J Biol Chem 277(47):45306-45314。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Sugiyama, K. and Nagata, K. (2016). Purification and Identification of Novel Host-derived Factors for Influenza Virus Replication from Human Nuclear Extracts. Bio-protocol 6(18): e1934. DOI: 10.21769/BioProtoc.1934; Full Text
  2. Sugiyama, K., Kawaguchi, A., Okuwaki, M. and Nagata, K. (2015). pp32 and APRIL are host cell-derived regulators of influenza virus RNA synthesis from cRNA. Elife 4: e08939.




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