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Expression and Purification of a Mammalian P2X7 Receptor from Sf9 Insect Cells
在Sf9昆虫细胞中表达纯化哺乳动物P2X7受体蛋白   

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eLIFE
Dec 2016

Abstract

The P2X7 receptor is an extracellular ATP-gated ion channel found only in eukaryotes (Bartlett et al., 2014). Due to its unique properties among P2X receptors, such as formation of a large conductance pore, the P2X7 receptor has been implicated in devastating diseases like chronic pain (North and Jarvis, 2013). However, mechanisms underlying the P2X7 specific properties remain poorly understood, partly because purification of this eukaryotic membrane protein has been challenging. Here we describe a detailed protocol for expressing and purifying a mammalian P2X7 receptor using an insect cell-baculovirus system. The P2X7 receptor is expressed in Sf9 insect cells as a GFP fusion protein and solubilized with a buffer containing Triton X-100 detergent. The P2X7-GFP fusion protein is then purified in a buffer containing dodecyl maltoside using Strep-Tactin affinity chromatography. Following enzymatic cleavage of the attached GFP and Strep-tag by thrombin, the P2X7 receptor is isolated using size exclusion chromatography. This method typically yields ~2 mg of purified protein from 6 L of Sf9 culture. Purified protein can be stored in a buffer containing 15% glycerol at 4 °C for at least 2 months and used for a variety of functional and structural studies (Karasawa and Kawate, 2016).

Keywords: P2X7 (P2X7), Sf9 (SF9), Baculovirus expression system (杆状病毒表达系统), Eukaryotic membrane protein (真核膜蛋白)

Background

The P2X7 receptor is one of the seven subtypes of the purinergic P2X receptor family and has been a promising novel drug target for a wide range of diseases such as neurodegenerative disorders, epilepsy, and neuropathic pain (North and Jarvis, 2013; Bhattacharya and Biber, 2016). Despite the well-documented clinical relevance, mechanisms underlying P2X7 specific functions are unclear. For example, it remains controversial whether P2X7 itself converts into a large pore or if P2X7 activation leads to an opening of another large-conductance channel such as pannexin1 (Alves et al., 2014).To unambiguously unravel the P2X7 receptor specific mechanisms, it is desirable to investigate the properties of this membrane channel in vitro in the absence of other proteins. It is also extremely advantageous to capture snapshots of the P2X7 receptor conformations throughout its gating cycle using techniques like X-ray crystallography and cryo-electron microscopy. However, purification of eukaryotic membrane proteins is nontrivial due to low expression levels and instability in detergents. Furthermore, complex folding mechanisms and necessary post-translational modifications force researchers to use eukaryotic host cells, which is time and cost consuming. Though several laboratories have established their own protocols for purifying eukaryotic membrane proteins using insect cells (Karakas et al., 2011; Hattori and Gouaux, 2012), it is often challenging to mimic experimental conditions simply based on the methods reported in research papers, which normally lack tips and special notes due to space limitation. This protocol aims to provide an in-depth guide for expressing and purifying eukaryotic membrane proteins using an insect cell/baculovirus system. Based on this protocol, we have successfully purified milligram quantities of a mammalian P2X7 receptor, which we used to determine its crystal structures (Karasawa and Kawate, 2016).

Materials and Reagents

  1. Generic pipette tips (VWR, catalog numbers: 613-0741 , 613-2133 , 613-0746 )
  2. Petri dish, 100 x 15 mm sterile disposable, polystyrene (VWR, catalog number: 470175-016)
    Manufacturer: Akro-Mils/Myers Industries, catalog number: 2900 .
  3. Conical bottom 15 ml and 50 ml polypropylene tubes (Greiner Bio One International, catalog numbers: 188280 , 227270 )
  4. Tube, with snap-on cap, polypropylene, 1.5 ml, 11 x 38 mm (Beckman Coulter, catalog number: 357448 )
  5. Neptune Microcentrifuge Tubes with Attached Flat Caps1.5 ml, 2 ml (Biotix, catalog numbers: 4445.X , 3765.X )
  6. Greiner CELLSTAR multi well culture plates 6 wells (TC treated with lid) (Greiner Bio One International, catalog number: 657160 )
  7. Syringe filter PVDF 0.22 μm 13 mm diameter (CELLTREAT Scientific Products, catalog number: 229742 )
  8. Corning 150 ml vacuum filter/storage bottle system, 0.22 µm, sterile (Corning, catalog number: 431154 )
  9. Amicon Ultra-4 Centrifugal Filter Units MWCO 100 kDa (EMD Millipore, catalog number: UFC810024 )
  10. Superdex 200 increase 10/300 GL (GE Healthcare, catalog number: 28990944 )
  11. Syringe (BD, catalog numbers: 329464 and 305559 )
  12. CELLSTAR Serological Pipettes 2, 5, 10, 25, 50 ml (Greiner Bio One International, catalog numbers: 710107 , 606107 , 607107 , 760107 , 768180 )
  13. Sf9 cells (Thermo Fisher Scientific, GibcoTM, catalog number: 11496015 )
  14. pNGFP-FB3 vector (developed in the Kawate lab) harboring a P2X7 receptor gene
    Note: In this protocol, panda P2X7 gene (NCBI Reference Sequence: XP_002913164.2) is used.
  15. MAX Efficiency DH5α competent cells (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18258012 )
  16. MAX Efficiency DH10Bac competent cells (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10361012 )
  17. Liquid nitrogen (Airgas, catalog number: NI 180LT230 )
  18. Nitrogen gas (Airgas, catalog number: NI HP300 )
  19. Sf-900 III serum free media (Thermo Fisher Scientific, GibcoTM, catalog number: 12658027 )
  20. SOC medium (Quality Biological, catalog number: 340-031-671 )
  21. E.Z.N.A. Plasmid Mini Kit (Omega Bio-tek, catalog number: D6942-02 )
  22. Phenol-chloroform (Sigma-Aldrich, catalog number: 77618 )
  23. Chloroform (Avantor Performance Materials, J.T. Baker®, catalog number: 9257-02 )
  24. 100% ethanol (Decon Labs, catalog number: V1016TP )
  25. HyClone penicillin-streptomycin 100x solution (GE Healthcare, HyCloneTM, catalog number: SV30010 )
  26. StrepTactin Sepharose High Performance (GE Healthcare, catalog number: 28-9355-99 )
  27. Human-Thrombin (Haematologic Technologies, catalog number: HCT-0020 )
  28. Cellfectin II (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10362100 )
  29. FuGENE 6 (Promega, catalog number: E2691 )
  30. jetPRIME (Polyplus-transfection, catalog number: 114-15 )
  31. Difco agar granulated (BD, DifcoTM, catalog number: 214530 )
  32. Bacto tryptone (BD, BactoTM, catalog number: 211705 )
  33. Bacto yeast extract (BD, BactoTM, catalog number: 212750 )
  34. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S641-212 )
  35. Kanamycin sulfate (Thermo Fisher Scientific, GibcoTM, catalog number: 15160054 )
  36. Gentamicin (Thermo Fisher Scientific, GibcoTM, catalog number: 15710064 )
  37. Tetracycline (Sigma-Aldrich, catalog number: 87128 )
  38. Bluo-gal (Teknova, catalog number: B1210 )
  39. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (EMD Millipore, catalog number: 420322 )
  40. Polyethylenimine (Polysciences, catalog number: 23966-1 )
  41. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: BP359-212 )
  42. Phosphate buffered saline (PBS) (Fisher Scientific, catalog number: BP399-20 )
  43. Leupeptin hemisulfate salt (Sigma-Aldrich, catalog number: L2884 )
  44. Aprotinin (Geno Technology, G-Bioscience, catalog number: 786-046 )
  45. Pepstatin (Enzo Life Sciences, catalog number: ALX-260-085-M100 )
  46. Phenylmethylsulfonyl fluoride (EMD Millipore, catalog number: 52332-25G )
  47. Triton X-100 (Anatrace, catalog number: T1001 )
  48. Tris-base (VWR, catalog number: 97061-794 )
  49. EDTA (Sigma-Aldrich, catalog number: EDS )
  50. n-Dodecyl-β-D-Maltopyranoside (Anatrace, catalog number: D310 )
  51. Hydrochloric acid (HCl) (VWR, BDH®, catalog number: BDH7204-4 )
  52. d-Desthiobiotin (Sigma-Aldrich, catalog number: D1411 )
  53. Glycerol (Alfa Aesar, catalog number: A16205 )
  54. LB-Bac plate (see Recipes)
  55. Polyethylenimine (see Recipes)
  56. Resuspension buffer (see Recipes)
  57. Solubilization buffer (see Recipes)
  58. Washing buffer (see Recipes)
  59. Elution buffer (see Recipes)
  60. SEC buffer (see Recipes)

Equipment

  1. Baker SterilGARDR II Biological safety Cabinet SG-600 (The Baker, model: Baker SterilGARDR II SG 600 )
  2. Isotemp digital-control water baths (Fisher Scientific, model: Model 2310 )
  3. Corning 250 ml polycarbonate Erlenmeyer flask with vent cap (Corning, catalog number: 431144 )
  4. Innova 44 shaker (Eppendorf, New BrunswickTM, model: Innova® 44 , catalog number: M1282-0000)
  5. Hemocytometer (Daigger Scientific, catalog number: EF16034F )
  6. Vortex Genie 2 (Scientific Industries, model: Vortex Genie 2 , catalog number: SI-0236)
  7. Evosf1 microscope (10x objective at 40% gain)
  8. 1 L Bottle with screw-on cap, polypropylene, 97 x 167 mm (Beckman Coulter, catalog number: 355676 )
  9. JS-4.2 rotor (Beckman Coulter, model: JS-4.2 , catalog number: 339080)
  10. Bottle assembly, polypropylene, 250 ml, 62 x 120 mm (Beckman Coulter, catalog number: 356011 )
  11. JA-14 rotor (Beckman Coulter, model: JA-14 , catalog number: 339247)
  12. Original Pipet-Aid pipette controller (DRUMMOND Scientific, catalog number: 4-000-110 )
  13. 4635 Cell disruption vessel (Parr Instrument, model: 4635 Cell Disruption Vessel )
  14. Bottle, assembly, polycarbonate, 70 ml, 38 x 102 mm, 1-1/2 x 4 in, Aluminum Cap (Beckman Coulter, catalog number: 355622 )
  15. Type 45 Ti rotor (Beckman Coulter, model: Type 45 Ti , catalog number: 339160)
  16. Thomas pestle tissue grinder assemblies with serrated pestles 10 ml (Thomas Scientific, catalog number: 3431E15 )
  17. 500 ml plastic beaker
  18. NanoDrop 2000 (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 , catalog number: ND-2000)
  19. Centrifuge 5810R (Eppendorf, model: 5810 R , catalog number: 5811000428)
  20. Centrifuge 5424 (Eppendorf, model: 5424 , catalog number: 022620401)
  21. J6-MI High-capacity centrifuge (Beckman Coulter, model: J6-MI , catalog number: 360291)
  22. Adaptor (Beckman Coulter, catalog number: 356096 )
  23. Avanti J-25I floor-model, refrigerated centrifuge (Beckman Coulter, model: Avanti J-25I ,catalog number: 363106)
  24. Optima LE-80K (Beckman Coulter, model: OptimaTM LE-80K , catalog number: 365668)
  25. Optima TLX (Beckman Coulter, model: OptimaTM TLX , catalog number: 361544)
  26. TLA-100.3 Rotor (Beckman Coulter, model: TLA-100.3 , catalog number: 349481)
  27. Adapter, delrin, tube, 11 mm dia (Beckman Coulter, catalog number: 355919 )
  28. AKTA purifier (GE Healthcare, model: ÄKTApurifier 10 , catalog number: 28406264)
  29. Econo-Pac chromatography columns (Bio-Rad Laboratories, catalog number: 7321010EDU )
  30. End caps, micro Bio-Spin chromatography columns (Bio-Rad Laboratories, catalog number: 7311660EDU )
  31. Mettler Toledo–XP204–Analytical Balance (Mettler-Toledo International, model: XP204S/M , catalog number: 11130054)
  32. OHAUS Harvard Trip Balances (OHAUS, catalog number: 80000005 )
  33. Milli-Q Reference Water Purification System (EMD Millipore, catalog number: Z00QSV0WW )
  34. SevenEasy pH meter (Mettler-Toledo International, catalog number: 51302819 )
  35. Corning 500 ml polycarbonate Erlenmeyer flask with vent cap (Corning, catalog number: 431145 )
  36. Corning 2 L polycarbonate Erlenmeyer flask with vent cap (Corning, catalog number: 431255 )
  37. 5 x 7 Inch Top PC-420D stirring plate (Corning, catalog number: 6798-420D )
  38. Magnetic stir bars
  39. Gravity and vacamatic sterilizers Amsco EAGLE SERIES 3000 (AMSCO, catalog number: 213415 )
  40. Fraction collector Frac-950 (GE Healthcare, model: Frac-950, catalog number: 18608300 )
  41. Refrigerator (Panasonic Biomedical, model: MPR-1411 )
  42. Steadystir digital S56 (Fisher Scientific, catalog number: 14-359-756 ) or an equivalent homogenizer
    Note: This product has been discontinued.
  43. F-500BAF, Ice maker (Hoshizaki America, model: F-500BAF )
  44. Globe Scientific Ice Bucket with Lid (Globe scientific, catalog number: IBB003P )
  45. C1000 Touch Thermal cycler (Bio-Rad Laboratories, catalog number: 1851148 )
  46. EVOS FL Imaging System (Thermo Fisher Scientific, catalog number: AMF4300 )
  47. EVOS Light Cube, GFP (Thermo Fisher Scientific, catalog number: AMEP4651 )

Software

  1. Vector NTI software 11.5 (Thermo Fisher Scientific)

Procedure

  1. Sf9 culture (perform in a tissue culture hood except for centrifugation steps)
    1. Sf9 cells in a cryo-vial are stored in liquid nitrogen.
    2. Thaw an aliquot (1-2 ml) of stock Sf9 insect cells (stored in liquid nitrogen) in a 37 °C water bath and resuspend into 10 ml Sf-900 III medium.
    3. Spin down the cells at 125 x g for 5 min, remove the medium by pipetting, and gently resuspend the cells in a 15 ml conical tube with 10 ml Sf900 III medium by pipetting up and down.
    4. Transfer the cells into a 250 ml flask and add 15 ml of Sf900 III to make a 25 ml cell suspension.
    5. Incubate the cells in a temperature controlled shaker at 27 °C and at 125 rpm.
    6. Monitor the cell density every day by manually counting the number of cells using a hemocytometer.
    7. When the cell density reaches 3.0-3.5 x 106 cells/ml (normally within 24-48 h), split the cells with Sf-900 III medium to make another 20 ml culture at 0.7 x 106 cells/ml.
    8. Incubate the cells in a temperature controlled shaker at 27 °C and at 125 rpm.
    9. After repeating the steps A6-A8 for 2-3 times, the doubling time should become approximately 24 h. Maintain Sf9 cultures at 0.5-3.5 x 106 cell/ml in a 250 ml flask and grow up in a larger flask for P2 virus infection.

  2. Preparation of bacmids
    1. The pNGFP-FB3 vector (Figure 1) harbors STREP tag, EGFP, thrombin recognition site, multiple cloning site (MCS) and a stop codon. The P2X7 receptor gene is subcloned between BamHI and XhoI sites.



      Figure 1. Vector map of pNFGP-FB3. This figure is created using Vector NTI software (Thermo Fisher Scientific).

    2. Incubate 25 μl of DH10Bac competent cells with ~100-250 ng of pNGFP-FB3[P2X7] on ice for 30 min.
    3. Heat shock the cells for 45 sec at 42 °C and put them on ice for 2 min.
    4. Add 500 μl of SOC medium into the cells and shake in a temperature controlled incubator at 225 rpm at 37 °C for 4 h.
    5. Harvest the cells by centrifugation at 20,000 x g for 1 min and plate them on a LB-Bac plate (see Recipes) (add ~50 μl SOC medium if necessary).
    6. Incubate the LB-Bac plate for 2 days at 37 °C.
    7. Pick up a white colony (Figure 2), inoculate 8 ml of LB-Bac medium, and culture in a temperature controlled incubator at 225 rpm at 37 °C overnight.


      Figure 2. Colonies of DH10Bac transformants. The picture was taken 2 days after transformation with the pNFGP-FB3-P2X7 construct.

    8. Harvest the cells by centrifugation at 2,500 x g for 10 min.
    9. Resuspend the cells with 200 μl of ‘Solution 1’ in Plasmid mini kit (Omega Bio-Tek).
    10. Add 200 μl of ‘Solution 2’ in Plasmid mini kit (Omega Bio-Tek) and mix by flipping the tube several times.
    11. Add 270 μl of ‘Solution 3’ in Plasmid mini kit (Omega Bio-Tek) and mix by flipping the tube several times.
    12. Harvest the bacmid containing solution by centrifugation at 20,000 x g for 10 min (save supernatant) and place it into a new 1.5 ml tube.
    13. Add 700 μl of phenol-chloroform and mix well by vortexing the tube for a few seconds.
    14. Centrifuge at 20,000 x g for 2 min and transfer the upper layer (Figure 3) into a new 1.5 ml tube.


      Figure 3. Phase separation after centrifugation at step B14. The upper layer includes bacmids.

    15. Add 700 μl of chloroform and mix well by vortexing for a few seconds.
    16. Centrifuge at 20,000 x g for 2 min and transfer the upper layer into a 2.0 ml tube.
    17. Add 1.4 ml of 100% ethanol and mix well by vortexing for a few seconds.
    18. Chill the tube at -20 °C for 15 min.
    19. Harvest the bacmid by centrifugation at 20,000 x g for 15 min at 4 °C.
    20. Rinse the bacmid containing pellet with 1 ml 70% ethanol.
    21. Aspirate the 70% ethanol and dry the bacmid containing pellet in a tissue culture hood for 30 min.
    22. Resuspend the bacmid with 50 μl sterile Milli-Q water in a tissue culture hood. Store bacmids at -20 °C.

  3. Virus production
    1. Plate Sf9 cells at 0.4 x 106 cells/well into a 6-well tissue culture plate.
    2. Incubate the plate at 27 °C for 1 h (Figure 4).


      Figure 4. Sf9 cells in 6-well plate before transfection. Image was taken using an Evosf1 microscope (10x objective at 40% gain).

    3. In the meantime, mix 5 μl of bacmid solution, 10 μl of polyethylenimine solution (see Recipes), and 100 μl of Sf-900 III medium.
    4. Incubate the mixture for 15 min at room temperature and add to the wells of the previously plated 6-well plate by pipetting dropwise.
    5. Incubate the plate at 27 °C for 6-7 days. GFP fluorescence was observed 7 days after transfection using an Evosf1 microscope (10x objective; 10% gain; Ex: 470/22 Em: 525/50) (Figure 5).


      Figure 5. GFP fluorescence from Sf9 cells expressing GFP-P2X7. GFP fluorescence 7 days after transfection. Image was obtained using an Evosf1 microscope (10x objective; 10% gain; Ex: 470/22 Em: 525/50). Scale bar = 400 μm.

    6. Collect the P1 virus containing media and filter sterilize using a 0.22 μm filter. Store the P1 virus at 4 °C.
    7. Inoculate 200 ml of Sf9 cells at 1.0 x 106 cells/ml with 100 μl of P1 virus.
    8. Culture the cells in a temperature controlled incubator at 125 rpm for 3 days at 27 °C.
    9. Collect the P2 virus containing media by centrifugation at 2,500 x g for 10 min. Filter sterilize the P2 virus using a 0.22 μm filter and store at 4 °C.

  4. Expression of P2X7 in Sf9 cells
    1. Set up 1 L Sf9 culture at 0.5 x 106 cells/ml and let them grow for 3 days at 125 rpm at 27 °C.
    2. Split the 1 L culture into 6 L (final density at 0.5 x 106 cells/ml). Add 7.5 ml/L of penicillin-streptomycin and culture in a temperature controlled shaker for 3 days at 125 rpm at 27 °C.
    3. When the cell density reaches 4.0 x 106 cells/ml, infect the Sf9 cells with 30 ml/L P2 virus. Culture at 27 °C for 24 h.
    4. Shift the temperature from 27 °C to 18 °C and culture for another 48 h before harvesting.

  5. Purification of P2X7 (perform all the steps at 4 °C or on ice)
    1. Transfer the GFP-P2X7 expressing cells into 1 L centrifuge bottles and spin down at 2,000 x g for 10 min (JS-4.2 rotor).
    2. After removing the culture media by decanting, resuspend the cells with ~20 ml/bottle of the resuspension buffer (see Recipes). Pellet should be yellow/green (Figure 6). Combine and transfer the cell suspensions into a 250 ml centrifuge bottle.


      Figure 6. Cell pellet after centrifugation (step E2)

    3. Harvest the cells by centrifugation at 3,800 x g for 10 min (JA-14 rotor).
    4. Carefully remove the resuspension buffer using a pipette and resuspend the cells with 200 ml of a fresh resuspension buffer.
    5. Break the cells by nitrogen cavitation using a cell disruption vessel (600 psi for 20 min; see Videos 1 and 2). Vessel should be incubated at 4 °C after nitrogen loading.

      Video 1. Filling nitrogen gas into the vessel

      Video 2. Collecting the lysates after 20 min incubation

    6. Sediment cell debris by centrifugation at 12,700 x g for 10 min (JA-14 rotor).
    7. Transfer the supernatant into three 70 ml ultracentrifuge tubes and balance them using the resuspension buffer. It is critical to balance the centrifuge tubes precisely and to fill it up to the shoulder of the tubes (Figure 7).


      Figure 7. Cell lysates in the ultracentrifuge tubes (step E7). Two tubes need to be balanced precisely.

    8. Centrifuge at 185,000 x g for 1 h (Ti-45 rotor).
    9. Remove the supernatant, resuspend the membrane fraction (pellet, Figure 8) with 2 ml/tube PBS.


      Figure 8. Collected membrane fraction after ultracentrifugation (step E9)

    10. Transfer the membrane fraction into a Dounce homogenizer (total volume is 8-12 ml; see Video 3).

      Video 3. Transfer the membrane fraction into a Dounce homogenizer

    11. Set the homogenizer at ~900 rpm and homogenize the membrane fraction with five strokes going up and down (see Video 4).

      Video 4. Resuspension of the membrane fraction with a Dounce homogenizer

    12. Solubilize the homogenized membrane fraction with ~350 ml of the solubilization buffer (see Recipes) by stirring at 300 rpm for 1 h.
    13. Transfer the supernatant into six 70 ml ultracentrifuge tubes and balance them using the solubilization buffer.
    14. Centrifuge at 185,000 x g for 1 h (Ti-45 rotor).
    15. Pool the supernatant (Figure 9) into a 500 ml beaker. Add 6 ml of Strep-Tactin resin pre-equilibrated with the solubilization buffer, and stir at 200 rpm for 60 min.


      Figure 9. Separated soluble fraction after ultracentrifugation (step E15)

    16.  Harvest the resin by centrifugation at 2,500 x g for 5 min in a 50 ml tube and transfer into two 25 ml gravity columns.
    17. Wash the resin with 20 ml of the washing buffer (see Recipes) for each column.
    18.  Elute the GFP-P2X7 protein with elution buffer (see Recipes; Figure 10).


      Figure 10. GFP-P2X7 bound to Strep resin

    19. Concentrate the eluted GFP-P2X7 down to 1 ml using a 100 kDa-cutoff spin column. Measure the protein concentration using a spectrophotometer.
    20. Digest the GFP-P2X7 with thrombin (25:1 [w/w]) overnight. In the meantime, equilibrate a Superdex 200 column with the SEC buffer (see Recipes) using an FPLC.
    21. Remove aggregated protein by centrifugation at 264,360 x g for 10min.
    22. Inject the protein into the Superdex 200 column and collect the peak fractions.

Data analysis

Quality of the purified P2X7 receptor can be analyzed by SEC and SDS-PAGE (Figure 11). Figure 11A shows a representative SEC profile with a single P2X7 peak, suggesting that the purified P2X7 receptor is monodisperse. A representative SDS-PAGE gel image (Figure 11B) verifies the chemical purity of this sample.


Figure 11. Characterization of purified P2X7. Representative SEC profile (A) and a gel image (B) of purified P2X7.

Notes

  1. Even a slightly higher concentration of gentamicin may be too toxic to DH10Bac cells. Use it exactly at 6.7 μg/ml.
  2. Cellfectin II (Thermo Fisher Scientific: 10362100), FuGENE 6 (Promega: E2691), and jetPRIME (polyplus, 114-15) could be also used for bacmid transfection.
  3. P2 virus should be always prepared fresh. Noticeable reduction of P2X7 receptor expression is observed with the usage of more than one week old virus.
  4. Temperature shift from 27 °C to 18 °C increases the expression level of P2X7 by more than four fold.
  5. Overgrown cells (> 5.0 x 106 cells/ml) result in low infection. Sf9 cells should be maintained within 30 passages (about 2 months).
  6. ESF921 medium (Expression system: 96-001-01) can also be used for Sf9 cell culture.
  7. Inclusion of glycerol in both elution and SEC buffers is necessary for avoiding aggregation of P2X7.

Recipes

  1. LB-Bac plate (1 L)
    10 g tryptone
    5 g yeast extract
    5 g NaCl
    50 μg/ml kanamycin, 6.7 μg/ml gentamicin, 10 μg/ml tetracycline, 100 μg/ml Bluo-gal, and 40 μg/ml IPTG
    MilliQ H2O to make it 1 L
  2. Polyethylenimine
    Dissolve 1 g polyethylenimine in 900 ml MilliQ water
    Adjust the pH to 7.0 with NaOH
    Volume up to 1 L with MilliQ water
    Filter sterilize using a 0.22 μm filter and store at 4 °C
  3. Resuspension buffer
    1x PBS
    0.5 μg/ml leupeptin
    2 μg/ml aprotinin
    0.5 μg/ml pepstatin
    0.5 mM PMSF
  4. Solubilization buffer
    1x PBS
    2% TritonX-100
  5. Washing buffer
    100 mM Tris
    150 mM NaCl
    1 mM EDTA
    0.5 mM Dodecyl-maltoside
    Adjust pH to 8.0 with HCl
  6. Elution buffer
    100 mM Tris
    150 mM NaCl
    1 mM EDTA
    0.5 mM Dodecyl-maltoside
    2.5 mM d-Desthiobiotin
    15% glycerol
    Adjust pH to 8.0 with HCl
  7. SEC buffer
    50 mM Tris
    150 mM NaCl
    15% glycerol
    0.5 mM Dodecyl-maltoside
    Adjust pH to 7.4 with HCl

Acknowledgments

We thank K. Michalski and P. Nguyen for critical comments. This protocol was adapted from our previous work (Karasawa and Kawate, 2016). This work was supported by the National Institutes of Health (GM114379 and NS072869).

References

  1. Alves, L. A., de Melo Reis, R. A., de Souza, C. A., de Freitas, M. S., Teixeira, P. C., Neto Moreira Ferreira, D. and Xavier, R. F. (2014). The P2X7 receptor: shifting from a low- to a high-conductance channel - an enigmatic phenomenon? Biochim Biophys Acta 1838(10): 2578-2587.
  2. Bartlett, R., Stokes, L. and Sluyter, R. (2014). The P2X7 receptor channel: recent developments and the use of P2X7 antagonists in models of disease. Pharmacol Rev 66(3): 638-675.
  3. Bhattacharya, A. and Biber, K. (2016). The microglial ATP-gated ion channel P2X7 as a CNS drug target. Glia 64(10): 1772-1787.
  4. Hattori, M. and Gouaux, E. (2012). Molecular mechanism of ATP binding and ion channel activation in P2X receptors. Nature 485(7397): 207-212.
  5. Karakas, E., Simorowski, N. and Furukawa, H. (2011). Subunit arrangement and phenylethanolamine binding in GluN1/GluN2B NMDA receptors. Nature 475(7355): 249-253.
  6. Karasawa, A. and Kawate, T. (2016). Structural basis for subtype-specific inhibition of the P2X7 receptor. Elife 5.
  7. North, R. A. and Jarvis, M. F. (2013). P2X receptors as drug targets. Mol Pharmacol 83(4): 759-769.

简介

P2X7受体是仅在真核生物中发现的胞外ATP门控离子通道(Bartlett等,2014)。由于其P2X受体之间的独特性质,例如大电导孔的形成,P2X7受体已经涉及破坏性疾病如慢性疼痛(North和Jarvis,2013)。然而,P2X7特异性属性的机制仍然知之甚少,部分原因是纯化这种真核膜蛋白是一个挑战。在这里,我们描述了使用昆虫细胞 - 杆状病毒系统表达和纯化哺乳动物P2X7受体的详细方案。 P2X7受体在作为GFP融合蛋白的Sf9昆虫细胞中表达,并用含有Triton X-100洗涤剂的缓冲液溶解。然后使用Strep-Tactin亲和层析在含有十二烷基麦芽糖苷的缓冲液中纯化P2X7-GFP融合蛋白。在通过凝血酶酶切割连接的GFP和Strep-标签后,使用大小排阻色谱分离P2X7受体。该方法通常从6L的Sf9培养物产生约2mg的纯化蛋白质。纯化的蛋白质可以用含有15%甘油的缓冲液在4℃下储存至少2个月,并用于各种功能和结构研究(Karasawa和Kawate,2016)。
【背景】P2X7受体是嘌呤能P2X受体家族的七种亚型之一,并且是广泛疾病如神经退行性疾病,癫痫和神经性疼痛的有希望的新型药物靶点(North和Jarvis,2013; Bhattacharya和Biber, 2016)。尽管有相当明确的临床相关性,P2X7具体功能的机制尚不清楚。例如,P2X7本身是否转化为大孔,或者如果P2X7激活导致另一个大电导通道如pannexin1的开放仍然是有争议的(Alves等,2014)。为了明确解开P2X7受体特异性机制,在没有其他蛋白质的情况下体外研究该膜通道的性质是理想的。使用诸如X射线晶体学和低温电子显微镜的技术,在其门控周期中捕获P2X7受体构象的快照也是非常有利的。然而,由于低表达水平和洗涤剂中的不稳定性,真核细胞膜蛋白的纯化是不重要的。此外,复杂的折叠机制和必要的翻译后修改迫使研究人员使用真核宿主细胞,这是时间和成本消耗的。虽然几个实验室已经建立了自己的使用昆虫细胞纯化真核细胞膜蛋白的方案(Karakas等人,2011; Hattori和Gouaux,2012),但是简单地根据研究论文中报道的方法来模拟实验条件往往具有挑战性,由于空间限制,通常缺少提示和特别说明。该方案旨在提供使用昆虫细胞/杆状病毒系统表达和纯化真核细胞膜蛋白的深入指南。基于该方案,我们已经成功地纯化了毫克量的哺乳动物P2X7受体,我们用于确定其晶体结构(Karasawa和Kawate,2016)。

关键字:P2X7, SF9, 杆状病毒表达系统, 真核膜蛋白

材料和试剂

  1. 通用移液器吸头(VWR,目录号:613-0741,613-2133,613-0746)
  2. 培养皿,100 x 15毫米无菌一次性聚苯乙烯(VWR,目录 号码:470175-016)
    制造商:Akro-Mils / Myers Industries,目录号:2900。
  3. 锥形底部15毫升 和50ml 聚丙烯管(Greiner Bio One 国际,目录号:188280, 227270)
  4. 管,带卡扣帽,聚丙烯,1.5毫升,11×38毫米(Beckman 库尔特,目录号:357448)
  5. 海王星微量离心管,附有平底帽,1.5毫升,2毫升(Biotix, 目录号:4445.X,3765.X)
  6. Greiner CELLSTAR多孔培养板6孔(用盖处理TC) (Greiner Bio One International,目录号:657160)
  7. 注射器过滤器PVDF 0.22μm13 mm直径(CELLTREAT Scientific 产品目录号:229742)
  8. 康宁150毫升真空过滤器/储存瓶系统,0.22微米,无菌 (康宁,目录号:431154)
  9. Amicon Ultra-4离心过滤器MWCO 100 kDa(EMD Millipore, 目录号:UFC810024)
  10. Superdex 200增加10/300 GL(GE Healthcare,目录 编号:28990944)
  11. 注射器(BD,目录号:329464和305559)
  12. 血清学 移液器2,5,10,25,50 ml(Greiner Bio One International,目录号:710107,606107,607107,760107,768180)
  13. Sf9细胞(Thermo Fisher Scientific,Gibco ,atalog号:11496015)
  14. pNGFP-FB3载体(在Kawate实验室开发)含有P2X7受体 基因
    注意:在本协议中,熊猫P2X7基因(NCBI参考序列: XP_002913164.2)。
  15. MAX效率DH5α感受态细胞(Thermo Fisher Scientific,Invitrogen TM, 目录号:18258012)
  16. MAX效率DH10Bac感受态细胞(Thermo Fisher Scientific,Invitrogen TM, 目录号:10361012)
  17. 液氮(Airgas,目录号:NI 180LT230)
  18. 氮气(Airgas,目录号:NI HP300)
  19. Sf-900 III血清游离培养基(Thermo Fisher Scientific,Gibco TM, 目录号:12658027)
  20. SOC介质(质量生物,目录号:340-031-671)
  21. E.Z.N.A. Plasmid Mini Kit(Omega Bio-tek,目录号:D6942-02)
  22. 苯酚氯仿(Sigma-Aldrich,目录号:77618)
  23. 氯仿(Avantor Performance 材料,J.T.贝克®,目录号:9257-02)
  24. 100%乙醇(Decon 实验室,目录号:V1016TP)
  25. HyClone青霉素 - 链霉素100x溶液(GE Healthcare,HyClone TM,目录号:SV30010)
  26. 链球菌素Sepharose 高性能(GE Healthcare,目录号:28-9355-99)
  27. 人血凝素(Haematologic Technologies,目录号:HCT-0020)
  28. Cellfectin II(Thermo Fisher Scientific,Invitrogen TM,目录 编号:10362100)
  29. FuGENE 6(Promega,目录号:E2691)
  30. jetPRIME(Polyplus转染,目录号:114-15)
  31. Difco琼脂造粒(BD,Difco TM,目录号:214530)
  32. Bacto胰蛋白胨(BD,Bacto TM ,目录号:211705)
  33. 细菌酵母提取物(BD,Bacto TM,目录号:212750)
  34. 氯化钠(NaCl)(Fisher Scientific,目录号:S641-212)
  35. 硫酸卡那霉素(Thermo Fisher Scientific,Gibco TM,目录 编号:15160054)
  36. 庆大霉素(Thermo Fisher Scientific,Gibco TM ,目录 编号:15710064)
  37. 四环素(Sigma-Aldrich,目录号:87128)
  38. Bluo-gal(Teknova,目录号:B1210)
  39. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(EMD Millipore,目录 编号:420322)
  40. 聚乙烯亚胺(Polysenences,目录号:23966-1)
  41. 氢氧化钠(NaOH)(Fisher Scientific,目录号:BP359-212)
  42. 磷酸盐缓冲盐水(PBS)(Fisher Scientific,目录号:BP399-20)
  43. 亮抑素半硫酸盐(Sigma-Aldrich,目录号:L2884)
  44. 抑肽酶(Geno Technology,G-Bioscience,目录号:786-046)
  45. Pepstatin(恩佐生命科学,目录号:ALX-260-085-M100)
  46. 苯基甲基磺酰氟(EMD Millipore,目录号:52332-25G)
  47. Triton X-100(Anatrace,目录号:T1001)
  48. 三碱基(VWR,目录号:97061-794)
  49. EDTA (Sigma-Aldrich,目录号:EDS)
  50. 正十二烷基-β-D-麦芽糖苷(Anatrace,目录号: D310)
  51. 盐酸(HCl)(VWR,BDH ,目录号:BDH7204-4)
  52. 雌二醇生物素(Sigma-Aldrich,目录号:D1411)
  53. 甘油(Alfa Aesar,目录号:A16205)
  54. LB-Bac板(见 食谱)
  55. 聚乙烯亚胺(见食谱)
  56. 再悬浮缓冲液(见 食谱)
  57. 溶解缓冲液 (见配方)
  58. 洗涤缓冲液(见 食谱)
  59. 洗脱缓冲液(见 食谱)
  60. SEC缓冲(见配方)

设备

  1. Baker SterilGARDR II生物安全柜SG-600(贝克,型号:Baker SterilGARDR II SG 600)
  2. Isotemp数字控制水浴(Fisher Scientific,型号:2310型)
  3. Corning 250ml聚碳酸酯锥形瓶,带通风孔(Corning,目录号:431144)
  4. Innova 44摇床(Eppendorf,New Brunswick TM ,型号:Innova ® 44,目录号:M1282-0000)
  5. 血细胞计数器(Daigger Scientific,目录号:EF16034F)
  6. Vortex Genie 2(Scientific Industries,型号:Vortex Genie 2,目录号:SI-0236)
  7. Evosf1显微镜(10倍物镜,40%增益)
  8. 1升带螺旋盖的聚丙烯,97 x 167 mm(Beckman Coulter,目录号:355676)
  9. JS-4.2转子(Beckman Coulter,型号:JS-4.2,目录号:339080)
  10. 瓶组件,聚丙烯,250毫升,62×120毫米(Beckman Coulter,目录号:356011)
  11. JA-14转子(Beckman Coulter,型号:JA-14,目录号:339247)
  12. 原始吸管辅助移液器控制器(DRUMMOND Scientific,目录号:4-000-110)
  13. 4635细胞破碎容器(Parr仪器,型号:4635细胞破碎容器)
  14. 瓶,组件,聚碳酸酯,70ml,38×102mm,1-1 / 2×4in,铝盖(Beckman Coulter,目录号:355622)
  15. 45型Ti转子(Beckman Coulter,型号:45 Ti,目录号:339160)
  16. 托马斯杵组织研磨机组件与锯齿杵10毫升(托马斯科学,目录号:3431E15)
  17. 500毫升塑料烧杯
  18. NanoDrop 2000(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM,目录号:ND-2000)
  19. 离心机5810R(Eppendorf,型号:5810 R,目录号:5811000428)
  20. 离心机5424(Eppendorf,型号:5424,目录号:022620401)
  21. J6-MI大容量离心机(Beckman Coulter,型号:J6-MI,目录号:360291)
  22. 适配器(Beckman Coulter,目录号:356096)
  23. Avanti J-25I型冷藏式离心机(Beckman Coulter,型号:Avanti J-25I,目录号:363106)
  24. Optima LE-80K(Beckman Coulter,型号:Optima TM LE-80K,目录号:365668)
  25. Optima TLX(Beckman Coulter,型号:Optima TM TLX,目录号:361544)
  26. TLA-100.3转子(Beckman Coulter,型号:TLA-100.3,目录号:349481)
  27. 适配器,delrin,管,11毫米直径(Beckman Coulter,目录号:355919)
  28. AKTA净化器(GE Healthcare,型号:ÄKTApurifier10,目录号:28406264)
  29. Econo-Pac色谱柱(Bio-Rad Laboratories,目录号:7321010EDU)
  30. 端帽,微生物旋转色谱柱(Bio-Rad Laboratories,目录号:7311660EDU)
  31. Mettler Toledo-XP204-分析天平(Mettler-Toledo International,型号:XP204S / M,目录号:11130054)
  32. OHAUS哈佛旅行余额(OHAUS,目录号:80000005)
  33. Milli-Q参考净水系统(EMD Millipore,目录号:Z00QSV0WW)
  34. SevenEasy pH计(Mettler-Toledo International,目录号:51302819)
  35. 康宁500ml聚碳酸酯锥形瓶,带通风孔(Corning,目录号:431145)
  36. Corning 2 L聚碳酸酯锥形瓶带通风孔(Corning,目录号:431255)
  37. 5 x 7英寸顶级PC-420D搅拌板(康宁,目录号:6798-420D)
  38. 磁力搅拌棒
  39. 重力和空气灭菌器Amsco EAGLE SERIES 3000(AMSCO,目录号:213415)
  40. 馏分收集器Frac-950(GE Healthcare,型号:Frac-950,目录号:18608300)
  41. 冰箱(Panasonic Biomedical,型号:MPR-1411)
  42. Steadystir数字S56(Fisher Scientific,目录号:14-359-756)或等效均质器
    注意:本产品已停产。
  43. F-500BAF,制冰机(Hoshizaki America,型号:F-500BAF)
  44. 地球科学冰桶盖(全球科学,目录号:IBB003P)
  45. C1000 Touch热循环仪(Bio-Rad Laboratories,目录号:1851148)
  46. EVOS FL成像系统(Thermo Fisher Scientific,目录号:AMF4300)
  47. EVOS Light Cube,GFP(Thermo Fisher Scientific,目录号:AMEP4651)

软件

  1. Vector NTI软件11.5(Thermo Fisher Scientific)

程序

  1. Sf9培养(除了离心步骤之外,在组织培养罩中进行)
    1. 冷冻瓶中的Sf9细胞储存在液氮中
    2. 在37℃水浴中解冻等份(1-2ml)的Sf9昆虫细胞(储存在液氮中),并重新悬浮于10ml Sf-900 III培养基中。
    3. 将细胞在125℃下旋转5分钟,通过移液去除培养基,并通过上下移液轻轻地将细胞悬浮在具有10ml Sf900 III培养基的15ml锥形管中。 >
    4. 将细胞转移到250ml烧瓶中,加入15ml Sf900III,制成25ml细胞悬液
    5. 在温度控制的振动器中,在27℃和125rpm下孵育细胞
    6. 通过使用血细胞计数器手动计数细胞数,每天监测细胞密度。
    7. 当细胞密度达到3.0-3.5×10 6细胞/ ml(通常在24-48小时内)时,用Sf-900Ⅲ培养基分裂细胞,在0.7×10 5细胞/ sup> 6 细胞/ ml。
    8. 在温度控制的振动器中,在27℃和125rpm下孵育细胞
    9. 在重复步骤A6-A8 2-3次后,加倍时间应变为约24小时。在250ml烧瓶中保持0.5-3.5×10 6细胞/ ml的Sf9培养物,并在较大的烧瓶中长成P2病毒感染。

  2. 杆菌的制备
    1. pNGFP-FB3载体(图1)含有STREP标签,EGFP,凝血酶识别位点,多克隆位点(MCS)和终止密码子。将P2X7受体基因亚克隆到BamHI和XhoI位点之间。



      图1. pNFGP-FB3的矢量图。该图是使用Vector NTI软件(Thermo Fisher Scientific)创建的。

    2. 在冰上孵育25μlDH10Bac感受态细胞〜100-250ng pNGFP-FB3 [P2X7] 30分钟。
    3. 在42℃下对细胞进行热休克45秒,并将其放在冰上2分钟
    4. 在细胞中加入500μlSOC培养基,并在温度控制的培养箱中以225rpm在37℃下振荡4小时。
    5. 通过以20,000×g离心收获细胞1分钟,并将其置于LB-Bac平板上(参见食谱)(如果需要,加入〜50μlSOC培养基)。
    6. 在37℃下孵育LB-Bac培养板2天。
    7. 拿起白色菌落(图2),接种8毫升LB-Bac培养基,并在温度控制的培养箱中以225rpm在37℃下培养过夜。


      图2. DH10Bac转化体的集落图片是用pNFGP-FB3-P2X7构建体转化后2天。

    8. 通过在2500Kg离心10分钟收获细胞。
    9. 在Plasmid mini试剂盒(Omega Bio-Tek)中用200μl"Solution 1"重悬细胞
    10. 在Plasmid迷你试剂盒(Omega Bio-Tek)中加入200μl"Solution 2",并将管子翻转数次。
    11. 在Plasmid迷你试剂盒(Omega Bio-Tek)中加入270μl"Solution 3",并将管子翻转数次。
    12. 通过以20,000×g离心10分钟(保存上清液)将含有杆粒的溶液收获并置于新的1.5ml管中。
    13. 加入700微升苯酚 - 氯仿,并将管子涡旋数秒钟混匀
    14. 以20,000 x g离心2分钟,并将上层(图3)转移到新的1.5 ml管中。


      图3.步骤B14离心后的相分离。上层包括杆状物。

    15. 加入700μl氯仿,涡旋混匀好几秒钟
    16. 以20,000 x g离心2分钟,并将上层转移到2.0ml管中。
    17. 加入1.4毫升100%乙醇,涡旋混匀好几秒钟
    18. 将管冷却至-20°C 15分钟
    19. 通过在4℃下以20,000×g离心15分钟收获杆粒。
    20. 用1ml 70%乙醇冲洗含有颗粒的颗粒
    21. 吸取70%乙醇,并在组织培养罩中干燥含有颗粒的粒细菌30分钟
    22. 用组织培养罩中的50μl无菌Milli-Q水重悬杆状杆菌。将杆菌储存在-20°C。

  3. 病毒生产
    1. 将平板Sf9细胞置于0.4×10 6细胞/孔中6孔组织培养板。
    2. 将板在27℃孵育1小时(图4)

      图4.转染前6孔板中的Sf9细胞使用Evosf1显微镜(10倍物镜,40%增益)拍摄图像。

    3. 同时,混合5μlbacmid溶液,10μl聚乙烯亚胺溶液(参见食谱)和100μlSf-900 III培养基。
    4. 将混合物在室温下孵育15分钟,并通过滴液加入先前镀6孔板的孔中。
    5. 将板在27℃孵育6-7天。使用Evosf1显微镜(10倍物镜; 10%增益; Ex:470/22 Em:525/50)(图5),转染后7天观察GFP荧光。

      图5.转染后7天的GFP荧光。表达GFP-P2X7的Sf9细胞的GFP荧光。 GFP荧光。使用Evosf1显微镜(10倍物镜; 10%增益; Ex:470/22 Em:525/50)获得图像。刻度棒=400μm。

    6. 收集含有P1病毒的培养基,并使用0.22μm过滤器进行过滤灭菌。将P1病毒存储在4°C。
    7. 用100μlP1病毒接种1.0×10 6个细胞/ ml的200ml Sf9细胞。
    8. 将细胞在温度控制的培养箱中以125rpm在27℃下培养3天
    9. 通过在2500Kg离心10分钟收集含有P2病毒的培养基。使用0.22μm过滤器对P2病毒进行过滤消毒,并在4°C储存
  4. P2X7在Sf9细胞中的表达
    1. 以0.5×10 6细胞/ ml设置1L Sf9培养物,并使其在27℃以125rpm生长3天。
    2. 将1L培养物分裂成6μL(最终密度为0.5×10 6个细胞/ ml)。加入7.5ml / L青霉素 - 链霉素,并在温度控制的振荡器中以27rpm在125rpm下培养3天。
    3. 当细胞密度达到4.0×10 6细胞/ ml时,用30ml / L P2病毒感染Sf9细胞。在27℃培养24小时。
    4. 将温度从27°C移至18°C,然后在收获前培养另外48小时。

  5. P2X7的纯化(在4°C或冰上执行所有步骤)
    1. 将GFP-P2X7表达细胞转移到1升离心瓶中,并以2,000×g旋转10分钟(JS-4.2转子)。
    2. 通过倾析除去培养基后,用〜20ml /瓶的重悬浮液重悬细胞(参见食谱)。颗粒应为黄色/绿色(图6)。将细胞悬浮液合并并转移到250ml离心瓶中

      图6.离心后的细胞沉淀(步骤E2)

    3. 通过在3,800×g离心10分钟(JA-14转子)收获细胞。
    4. 使用移液管小心地去除重悬浮液,并用200毫升新鲜的重悬浮液重新悬浮细胞。
    5. 使用细胞破碎容器(600psi,20分钟;见视频1和2),通过氮气蚀破坏细胞。 容器应在氮气负载后在4℃下孵育
      Video 1. Filling nitrogen gas into the vessel

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      Video 2. Collecting the lysates after 20 min incubation

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    6. 沉淀细胞碎片通过在12,700×g下离心10分钟(JA-14转子)。
    7. 将上清液转移到三个70 ml超离心管中,并使用再悬浮缓冲液进行平衡。将离心管精确平衡并将其填充到管的肩部至关重要(图7)

      图7.超离心管中的细胞裂解物(步骤E7)两根管需要精确平衡。

    8. 以185,000 x g离心1小时(Ti-45转子)。
    9. 取出上清液,用2 ml /管PBS重新悬浮膜级分(颗粒,图8)

      图8.超速离心后收集的膜级分(步骤E9)

    10. 将膜部分转移到Dounce匀浆器中(总体积为8-12ml;见视频3)
      Video 3. Transfer the membrane fraction into a Dounce homogenizer

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    11. Set the homogenizer at ~900 rpm and homogenize the membrane fraction with five strokes going up and down (see Video 4).

      Video 4. Resuspension of the membrane fraction with a Dounce homogenizer

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    12. 通过在300rpm下搅拌1小时,使约350ml溶解缓冲液(参见食谱)使均匀化的膜级分溶解。
    13. 将上清液转移到六个70 ml超离心管中,并使用溶解缓冲液进行平衡。
    14. 以185,000×g离心1小时(Ti-45转子)。
    15. 将上清液(图9)放入500ml烧杯中。加入6ml用增溶缓冲液预平衡的Strep-Tactin树脂,并以200rpm搅拌60分钟。


      图9.超离心后分离的可溶级分(步骤E15)

    16.                         
    17. 用20ml洗涤缓冲液洗涤树脂(参见食谱)。
    18. 用洗脱缓冲液洗脱GFP-P2X7蛋白质(见配方;图10)

      图10.与Strep树脂结合的GFP-P2X7

    19. 使用100 kDa截止的旋转柱将洗脱的GFP-P2X7浓缩至1 ml。使用分光光度计测量蛋白质浓度。
    20. 用凝血酶(25:1 [w / w])将GFP-P2X7消化过夜。同时,使用FPLC平衡Superdex 200色谱柱与SEC缓冲液(参见食谱)。
    21. 通过以264,360×g离心10分钟去除聚集的蛋白质。
    22. 将蛋白质注入Superdex 200柱,并收集峰分数。

数据分析

纯化的P2X7受体的质量可以通过SEC和SDS-PAGE进行分析(图11)。图11A显示具有单个P2X7峰的代表性SEC曲线,表明纯化的P2X7受体是单分散的。代表性的SDS-PAGE凝胶图像(图11B)验证了该样品的化学纯度

图11.纯化的P2X7的表征。代表性的SEC图谱(A)和纯化的P2X7的凝胶图像(B)。

笔记

  1. 即使是较高浓度的庆大霉素也可能对DH10Bac细胞毒性过大。使用它完全是6.7μg/ ml。
  2. Cellfectin II(Thermo Fisher Scientific:10362100),FuGENE 6(Promega:E2691)和jetPRIME(polyplus,114-15)也可用于杆粒转染。
  3. P2病毒应该总是准备新鲜。观察到P2X7受体表达的显着降低,使用了一周以上的病毒。
  4. 温度从27°C变化到18°C时,P2X7的表达水平提高了四倍以上
  5. 长满的细胞(> 5.0×10 6细胞/ ml)导致低感染。 Sf9细胞应保持在30代以内(约2个月)
  6. ESF921培养基(表达系统:96-001-01)也可用于Sf9细胞培养
  7. 在洗脱和SEC缓冲液中包含甘油对于避免P2X7的聚集是必要的。

食谱

  1. LB-Bac板(1升)
    10克的胰蛋白胨
    5克酵母提取物
    5克NaCl
    50μg/ ml卡那霉素,6.7μg/ ml庆大霉素,10μg/ ml四环素,100μg/ ml Bluo-gal和40μg/ ml IPTG
    MilliQ H< 2> O以使其成为1L
  2. 聚乙烯亚胺
    将1g聚乙烯亚胺溶于900毫升MilliQ水中 用NaOH调节pH至7.0 体积达1升,MilliQ水
    使用0.22μm过滤器进行过滤消毒,并在4°C下储存
  3. 再悬浮缓冲液
    1x PBS
    0.5μg/ ml亮抑酶肽
    2μg/ ml抑肽酶
    0.5μg/ ml胃蛋白酶抑制剂
    0.5 mM PMSF
  4. 溶解缓冲液
    1x PBS
    2%TritonX-100
  5. 洗涤缓冲液
    100 mM Tris
    150 mM NaCl
    1 mM EDTA
    0.5mM十二烷基麦芽糖苷 用HCl调节pH至8.0
  6. 洗脱缓冲液
    100 mM Tris
    150 mM NaCl
    1 mM EDTA
    0.5mM十二烷基麦芽糖苷 2.5mM d-生物素生物素
    15%甘油
    用HCl调节pH至8.0
  7. SEC缓冲区
    50 mM Tris
    150 mM NaCl
    15%甘油
    0.5mM十二烷基麦芽糖苷 用HCl调节pH至7.4

致谢

我们感谢K. Michalski和P. Nguyen的批评性意见。这个协议是从我们以前的工作(Karasawa和Kawate,2016)改编而成。这项工作得到了国家卫生研究院(GM114379和NS072869)的支持。

参考

  1. Alves,LA,de Melo Reis,RA,de Souza,CA,de Freitas,MS,Teixeira,PC,Neto Moreira Ferreira,D. and Xavier,RF(2014)。  P2X7受体:从低电导通道转移到高电导通道 - 一个神秘的现象? Biochim Biophys Acta 1838(10):2578-2587。
  2. Bartlett,R.,Stokes,L。和Sluyter,R。(2014)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/24928329"目标="_ blank"> P2X7受体通道:最近的发展和P2X7拮抗剂在疾病模型中的应用。 Pharmacol Rev 66(3):638-675。
  3. Bhattacharya,A.和Biber,K.(2016)。小胶质细胞ATP门控离子通道P2X7作为中枢神经系统药物靶标。 64(10):1772-1787。
  4. Hattori,M.和Gouaux,E.(2012)。 P2X受体中ATP结合和离子通道活化的分子机制。自然 485(7397):207-212。
  5. Karakas,E.,Simorowski,N。和Furukawa,H。(2011)。 GluN1 / GluN2B NMDA受体中的亚基排列和苯乙醇胺结合。自然475(7355):249-253。
  6. Karasawa,A.和Kawate,T。(2016)。 P2X7受体的亚型特异性抑制的结构基础 Elife 5.
  7. 北,RA和Jarvis,MF(2013)。 P2X受体作为药物靶标。 Mol Pharmacol 83(4):759-769。
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Copyright Karasawa and Kawate. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Karasawa, A. and Kawate, T. (2017). Expression and Purification of a Mammalian P2X7 Receptor from Sf9 Insect Cells. Bio-protocol 7(17): e2544. DOI: 10.21769/BioProtoc.2544.
  2. Karasawa, A. and Kawate, T. (2016). Structural basis for subtype-specific inhibition of the P2X7 receptor. Elife 5.
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