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Expression, Purification and Crystallisation of the Adenosine A2A Receptor Bound to an Engineered Mini G Protein
与改造的迷你G蛋白结合的腺苷A2A受体的表达、纯化和结晶   

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Abstract

G protein-coupled receptors (GPCRs) promote cytoplasmic signalling by activating heterotrimeric G proteins in response to extracellular stimuli such as light, hormones and nucleosides. Structure determination of GPCR–G protein complexes is central to understanding the precise mechanism of signal transduction. However, these complexes are challenging targets for structural studies due to their conformationally dynamic and inherently transient nature. We recently developed an engineered G protein, mini-Gs, which addressed these problems and allowed the formation of a stable GPCR–G protein complex. Mini-Gs facilitated the structure determination of the human adenosine A2A receptor (A2AR) in its G protein-bound conformation at 3.4 Å resolution. Here, we describe a step by step protocol for the expression and purification of A2AR, and crystallisation of the A2AR–mini-Gs complex.

Keywords: Adenosine A2A receptor(腺苷A2A受体), A2AR(A2AR), Active state(活性状态), GPCR(GPCR), G protein-coupled receptor(G蛋白偶联受体), Mini G protein(迷你G蛋白), Mini-Gs(迷你Gs), G protein complex(G蛋白复合物)

Background

We recently developed an engineered minimal G protein, mini-Gs (Carpenter and Tate, 2016), which facilitated the structure determination of the human adenosine A2A receptor (A2AR) in its active state (Carpenter et al., 2016). Mini-Gs stabilises the active conformation of A2AR sufficiently to allow crystallization of the complex by vapour diffusion in the detergent octylthioglucoside. Here, we describe a detailed protocol for the expression and purification of A2AR, which is adapted from a previously described method developed in our laboratory (Lebon et al., 2011a and 2011b; Tate and Lebon, 2015). We also describe a step by step procedure for the preparation and crystallisation of the A2AR–mini-Gs complex, earlier described in Carpenter et al. (2016). Expression and purification of mini-Gs is described in a companion manuscript (Carpenter and Tate, 2017).

Materials and Reagents

  1. Serological pipette
  2. Pipette tips (STARLAB INTERNATION)
  3. Plastic spatula
  4. 50 ml tubes (SARSTEDT, catalog number: 62.547.254 )
  5. 15 ml tubes (SARSTEDT, catalog number: 62.554.002 )
  6. 5 ml tubes (Eppendorf, catalog number: 0030119401 )
  7. 1.5 ml tubes (SARSTEDT, catalog number: 72.690.001 )
  8. 0.5 ml tubes (SARSTEDT, catalog number: 72.699 )
  9. Steritop 0.22 μm filter unit (EMD Millipore, catalog number: SCGPT01RE )
  10. Amicon Ultra-15 concentrator 50 kDa cut-off (EMD Millipore, catalog number: UFC905024 )
  11. Plastic column (e.g., empty PD-10 column) (GE Healthcare, catalog number: 17043501 )
  12. PD-10 desalting column (GE Healthcare, catalog number: 17085101 )
  13. Amicon Ultra-4 concentrator 50 kDa cut-off (EMD Millipore, catalog number: UFC805024 )
  14. MRC 96-well 2-drop crystallization plates (Molecular Dimensions, catalog number: MD11-00-100 )
  15. Ni2+-NTA Superflow 5 ml prepacked column (QIAGEN, catalog number: 30760 )
  16. Superdex 200 GL 10/300 gel filtration column (GE Healthcare, catalog number: 17517501 )
  17. Trichoplusia ni (T. ni) insect cell line (Expression Systems, catalog number: 94-002F )
  18. pBacPAK8 plasmid (Takara Bio, Clontech, catalog number: PT1262-5 )
  19. flashBAC ULTRA DNA (Oxford Expression Technologies, catalog number: 100300 )
  20. ESF921 insect cell media (Expression Systems, catalog number: 96-001-01 )
  21. Fetal bovine serum (Sigma-Aldrich, catalog number: F9665 )
  22. cOmplete, EDTA-free protease inhibitor tablets (Roche Diagnostics, catalog number: 11873580001 )
  23. PMSF (Sigma-Aldrich, catalog number: P7626 )
  24. Liquid nitrogen
  25. NECA (Sigma-Aldrich, catalog number: E2387 )
  26. Imidazole (Sigma-Aldrich, catalog number: 56748 )
  27. Sodium chloride (NaCl) (Fisher Scientific, catalog number: 10598630 )
  28. n-Decyl β-maltoside (DM) detergent (Anatrace, catalog number: D322 )
  29. TEV protease (produced in-house)
  30. Ni2+-NTA agarose (QIAGEN, catalog number: 30210 )
  31. Magnesium chloride (MgCl2) (Fisher Scientific, catalog number: BP214-500 )
  32. Apyrase (Sigma-Aldrich, catalog number: A6535 )
  33. Sodium acetate (Fisher Scientific, catalog number: 10794761 )
  34. MemGoldTM crystallisation screen (Molecular Dimensions, catalog number: MD1-39 )
  35. PEG 2000 (Sigma-Aldrich, catalog number: 81221 )
    Note: This product has been discontinued.
  36. PEG 2000 MME (Sigma-Aldrich, catalog number: 81321 )
    Note: This product has been discontinued.
  37. PEG 400 (Hampton Research, catalog number: HR2-603 )
  38. Glycerol (VWR, catalog number: 24388.320 )
  39. HEPES (Sigma-Aldrich, catalog number: H3375 )
  40. EDTA
  41. Absolute ethanol (VWR, catalog number: 20821.330 )
  42. DMSO (Sigma-Aldrich, catalog number: D2650 )
  43. n-Octyl-β-D-thioglucoside (OTG) detergent (Glycon, catalog number: D20014 )
  44. Precision Plus SDS-PAGE molecular weight standards (Bio-Rad Laboratories, catalog number: 161-0373 )
  45. 4-20% Tris-glycine SDS-PAGE gels (Fisher Scientific, catalog number: EC60255BOX )
  46. Buffer A (see Recipes)
  47. PMSF stock solution (see Recipes)
  48. NECA stock solution (see Recipes)
  49. DM stock solution (see Recipes)
  50. Buffer B (see Recipes)
  51. Buffer C (see Recipes)
  52. Buffer D (see Recipes)
  53. Buffer E (see Recipes)
  54. OTG stock solution (see Recipes)
  55. Apyrase stock solution (see Recipes)
  56. Buffer F (see Recipes)

Equipment

  1. Pipettes (STARLAB INTERNATION)
  2. Optimum GrowthTM 5 L flask (Thompson Instrument Company, catalog number: 931116 )
  3. Shaker incubator (Infors, model: Multitron Standard )
  4. High speed centrifuge (e.g., Beckman Coulter, model: Avanti J-26XP , catalog number: 393124)
  5. Type 45 Ti (Ti45) ultracentrifuge rotor (Beckman Coulter, catalog number: 339160 )
  6. Type 45 Ti (Ti45) ultracentrifuge bottle assembly (Beckman Coulter, catalog number: 355622 )
  7. Water bath (Julabo)
  8. ULTRA-TURRAX T25 homogeniser (IKA, model: ULTRA-TURRAX T25 Homogeniser )
  9. Magnetic stirring bar
  10. Type 70 Ti (Ti70) ultracentrifuge rotor (Beckman Coulter, catalog number: 337922 )
  11. Type 70 Ti (Ti70) ultracentrifuge bottle assembly (Beckman Coulter, catalog number: 355618 )
  12. Peristaltic pump (e.g., GE Healthcare, model: Pump P-1 , catalog number: 18-1110-91)
  13. Refrigerated benchtop centrifuge (e.g., Eppendorf, catalog number: 5430 R )
  14. Roller mixer (IKA, model: ROLLER 6 digital , catalog number: 0004011000)
  15. TLA55 benchtop ultracentrifuge rotor (Beckman Coulter, catalog number: 366725 )
  16. Optima L100 XP preparative ultracentrifuge (Beckman Coulter, model: OptimaTM L-100 XP )
  17. Optima MAX benchtop ultracentrifuge (Beckman Coulter, model: OptimaTM MAX )
  18. Refrigerated microcentrifuge (e.g., Eppendorf, catalog number: 5418 R )
  19. Rotor capable of spinning 1 L bottles (e.g., JLA-8.1000) (Beckman Coulter, catalog number: 363688 )
  20. ÄKTA Purifier chromatography system (GE Healthcare, model: ÄKTA Purifier )
  21. Mosquito® Crystal protein crystallisation robot (TTP Labtech, model: mosquito® Crystal )

Software

  1. UNICORN (GE Healthcare)
  2. Graphical software (e.g., Prism 7) (GraphPad)

Procedure

  1. Expression of A2AR in insect cells
    Human A2AR, which was truncated to remove the flexible C-terminus (see Note 1) and contained the N154A mutation to remove a potential N-linked glycosylation site, was cloned into the transfer vector pBacPAK8, and baculoviruses were prepared using the flashBAC ULTRA system, following the manufacturer’s instructions. The A2AR construct contained a C-terminal 10x histidine tag to facilitate purification (see Note 2) and a TEV protease cleavage site to allow removal of the histidine tag (Figure 1).


    Figure 1. A2AR baculovirus expression construct. A2AR was cloned into the baculovirus transfer vector pBacPAK8 using BamHI and NotI restriction sites (underlined), and baculoviruses were prepared using the flashBAC ULTRA system. The construct consists of residues 1-308 of human A2AR followed by a TEV protease cleavage site (highlighted in green) and C-terminal 10x histidine tag (highlighted in red). A2AR contains the N154A mutation (highlighted in yellow) to remove a potential N-linked glycosylation site. Start and stop codons are shown in bold.

    1. Dilute T. ni cells (grown in ESF921 serum-free media) to 2,730 ml in a 5 L optimum growth flask at a density of 1 x 106 cells/ml, and incubate overnight at 27 °C, shaking at 124 rpm.
    2. Once the density reaches 3.3 x 106 cells/ml, add 150 ml of fetal bovine serum (final concentration of 5% v/v) and 120 ml of passage three A2AR baculovirus (final concentration of 4% v/v).
    3. Incubate for 65-72 h at 27 °C, shaking at 124 rpm.
    4. Harvest the cells by centrifugation at 5,000 x g for 10 min at 4 °C.
    5. Resuspend the cell pellet to a final volume of 180 ml in buffer A, add 4 protease inhibitor tablets and PMSF to give a final concentration of 1 mM. Flash freeze in liquid nitrogen and store at -80 °C.

  2. Insect cell membrane preparation
    A crude membrane preparation is performed, which retains all insoluble intracellular components, whilst removing soluble proteins. This type of membrane preparation helps to limit the loss of membrane fragments and maximise the recovery of A2AR. This protocol describes the membrane preparation from three liters of insect cell culture, which is sufficient for one A2AR purification. However, if necessary, two batches (six liters of culture in total) can be processed simultaneously using a single Ti45 ultracentrifuge rotor.
    1. Thaw the cell pellet from 3 L of insect cell culture in a water bath at room temperature.
    2. Add fresh PMSF to give a final concentration of 1 mM, transfer the solution to three Ti45 tubes and centrifuge at 158,000 x g (45,000 rpm) in a Ti45 rotor for 2 h at 4 °C. Make sure the tubes are filled correctly (to the neck), top up with buffer A if necessary. (see Note 3)
    3. Carefully remove the supernatant until level with the top of the pellet using a serological pipette (see Figure 2).


      Figure 2. Illustration of the membrane pellet after ultracentrifugation. A diffuse layer, which contains a significant amount of A2AR, is observed on the surface of the membrane pellet after ultracentrifugation. It is important not to decant the supernatant, because this will remove the diffuse layer, resulting to a decreased yield of A2AR. Instead, the supernatant should be carefully aspirated using a serological pipette until level with the top of the membrane pellet (indicate by the dashed line).

    4. Add 25 ml of buffer A to each tube and break the pellet up into small pieces using plastic spatula.
    5. Homogenise the pellet using an ULTRA-TURRAX T25 homogeniser operating at 10,000 rpm until no lumps remain. This should take approximately 30 sec per tube.
    6. Top up the tubes with buffer A and centrifuge at 158,000 x g (45,000 rpm) in a Ti45 rotor for 2 h at 4 °C. (see Note 3)
    7. Remove the supernatant until level with the top of the pellet using a serological pipette (see Figure 2).
    8. Resuspend the pellet with the buffer that remains in the tubes using a plastic spatula followed by an ULTRA-TURRAX T25 homogeniser. Do not exceed a final volume of 160 ml. Aliquot the membrane suspension into 50 ml tubes, flash freeze in liquid nitrogen and store at -80 °C. Membranes can be stored under these conditions for up to three months.

  3. Purification of A2AR
    A2AR is purified in decylmaltoside (DM), in the presence of the agonist NECA, using a protocol adapted from a previously described method for the purification of a thermostabilised A2AR construct (Lebon et al., 2011a and 2011b; Tate and Lebon, 2015). DM must be used if the complex is to be later exchanged into short chain detergents for vapour diffusion crystallisation. This protocol typically yields 3-6 mg of pure A2AR from 3 L of insect cell culture.
    1. Thaw the membranes from 3 L of insect cell culture in a water bath at room temperature.
    2. Add 4 protease inhibitor tablets, and NECA (100 μM), imidazole (10 mM), PMSF (1 mM), and NaCl (300 mM) to give the final concentrations indicated, stir slowly at room temperature for 30 min using a magnetic stirring bar to allow agonist binding.
    3. Cool the membrane suspension to 4 °C in an ice bath. Solubilise the membranes by adding DM to give a final concentration of 2% (w/v) while stirring rapidly using a magnetic stirring bar, continue to stir slowly for 30 min at 4 °C.
    4. Transfer the solution to eight Ti70 tubes and centrifuge at 265,000 x g (60,000 rpm) in a Ti70 rotor for 2 h at 4 °C.
    5. Remove the supernatant and filter through a 0.2 μm Steritop filter unit. (see Note 4)
    6. Load the filtrate (~150 ml) onto a 5 ml Ni2+-NTA Superflow column (pre-equilibrated with buffer B) at a flow rate of 2 ml/min at 4 °C, using a peristaltic pump. The filtrate should be kept on ice during loading.
    7. Wash the column with 100 ml of buffer C at a flow rate of 5 ml/min at 4 °C.
    8. Elute the column with 20 ml of buffer D at a flow rate of 2 ml/min at 4 °C, collecting a single 20 ml fraction.
    9. Concentrate the eluate to 5 ml using an Amicon Ultra-15 concentrator (50 kDa molecular weight cut-off) in a refrigerated benchtop centrifuge at 4 °C. (see Note 5)
    10. Exchange the concentrated protein into buffer E using two PD-10 desalting columns (load 2.5 ml onto each column). (see Note 6)
    11. Add TEV protease to give a TEV:A2AR ratio of approximately 1:3 (w/w), incubate overnight on ice.
    12. Add 2 ml of Ni2+-NTA agarose resin (pre-equilibrated with buffer E), mix on a roller mixer for 30 min at 4 °C.
    13. Pour the suspension onto 1 ml of Ni2+-NTA agarose resin (pre-equilibrated with buffer E) packed into a disposable plastic column and let it run through by gravity.
    14. Collect the flow through and wash the column with 2 x 3 ml of buffer E.
    15. Pool the wash with the flow through and concentrate to 0.2 ml using an Amicon Ultra-15 concentrator (50 kDa cut-off) in a refrigerated benchtop centrifuge at 4 °C.
    16. Centrifuge the concentrated protein at 135,000 x g (55,000 rpm) in a TLA55 rotor using a benchtop ultracentrifuge for 10 min at 4 °C to remove aggregates.
    17. Load the supernatant onto a Superdex 200 GL 10/300 gel filtration column, (pre-equilibrated with buffer E) at a flow rate of 0.4 ml/min at 4 °C, collecting 0.5 ml fractions.
    18. Pool peak fractions (typically 3-4 ml) based on the gel filtration chromatogram. (see Note 7)
    19. Concentrate the pooled fractions to 0.2 ml using an Amicon Ultra-4 concentrator (50 kDa cut-off) in a refrigerated benchtop centrifuge at 4 °C.
    20. Determine the protein concentration using the amido black assay (Schaffner and Weissmann, 1973). (see Note 8)
    21. If required, purified A2AR can be aliquoted, flash frozen in liquid nitrogen and stored at -80 °C. (see Note 9) 

  4. Crystallisation of the A2AR–mini-Gs complex
    The A2AR–mini-Gs complex is first prepared in DM, once the complex is formed it is stable enough to be exchanged into octylthioglucoside (OTG) for vapour diffusion crystallisation trials. A detailed protocol for the expression and purification of mini-Gs is provided in a companion manuscript (Carpenter and Tate, 2017).
    1. Aliquot 2 mg of A2AR into a microcentrifuge tube and add a 1.2-fold molar excess of mini-Gs construct 414 (1.8 mg). Adjust the volume to 0.2 ml with buffer E, add MgCl2 to give a final concentration of 1 mM and add 0.1 units of apyrase. Incubate for 3-16 h on ice. (see Note 10)
    2. Start the detergent exchange (from DM to OTG) by diluting the complex 1:10 in buffer F, and re-concentrating to 0.2 ml using an Amicon Ultra-4 concentrator (50 kDa cut-off) in a refrigerated benchtop centrifuge at 4 °C.
    3. Centrifuge the concentrated protein at 55,000 rpm (135,000 x g) in a TLA55 rotor using a benchtop ultracentrifuge for 10 min at 4 °C to remove aggregates.
    4. Complete the detergent exchange by loading the supernatant onto a Superdex 200 GL 10/300 gel filtration column (pre-equilibrated with buffer F) at a flow rate of 0.5 ml/min at 4 °C, collecting 0.5 ml fractions.
    5. Pool peak fractions (typically 2-3 ml) based on the gel filtration chromatogram (Figure 3).


      Figure 3. SDS-PAGE and gel filtration analysis of the A2AR purification. A and B. SDS-PAGE analysis of the purification of a representative A2AR construct. Despite having molecular weights of 35 and 27 kDa respectively, A2AR and mini-Gs414 migrate at an identical rate on the SDS-PAGE gel, making visualisation of the complex difficult. Therefore, for demonstration purposes, we show SDS-PAGE gels of an A2AR construct that contains an N-terminal thioredoxin fusion (TrxA-A2AR), which increases its molecular weight to 46 kDa. TrxA-A2AR was purified in DM using an identical protocol to that described for A2AR, however the TrxA-A2AR–mini-Gs complex, which was exchanged into OTG, did not yield crystals that diffracted to higher than 3.5 Å resolution. A. SDS-PAGE analysis of the TrxA-A2AR purification: (M) molecular weight markers; (1) Ni2+-NTA column loading material (1:10 dilution); (2) Ni2+-NTA column flow through (1:10 dilution); (3) Ni2+-NTA column wash; (4) Ni2+-NTA column eluate; (5) sample after concentration and buffer exchange; (6) sample after TEV digestion; (7) Ni2+-NTA negative purification flow through; (8) Ni2+-NTA negative purification eluate; (9) gel filtration pool. TEV protease is indicated by an asterisk. The samples should not be boiled prior to loading on the gel (see Note 11). B. SDS-PAGE analysis of the TrxA-A2AR–mini-Gs414 complex after detergent exchange into OTG: (M) molecular weight markers; (1) gel filtration pool. C. Preparative gel filtration chromatogram for the A2AR–mini-Gs414 complex in OTG, pooled fractions (typically 2-3 ml) are indicated by dashed lines. Note that the UV detector reaches saturation, which results in a truncated absorbance profile. D. A crystal of the A2AR–mini-Gs414 complex mounted in a loop on beamline ID23-2 at the European Synchrotron Radiation Facility (the scale bar is 50 x 100 μm).

    6. Concentrate the pooled fractions to ≥ 20 mg/ml using an Amicon Ultra-4 concentrator (50 kDa cut-off) in a refrigerated benchtop centrifuge at 4 °C.
    7. Centrifuge the concentrated protein at 135,000 x g (55,000 rpm) in a TLA55 rotor using a benchtop ultracentrifuge for 10 min at 4 °C to remove aggregates.
    8. Transfer the supernatant to a new tube and discard the pellet.
    9. Accurately determine the concentration of the complex using the amido black assay (Schaffner and Weissmann, 1973).
    10. Dilute the complex to 20 mg/ml and immediately set up crystallisation trials in 96-well sitting drop plates, dispensing 120 nl of protein and 120 nl of precipitant solution using a Mosquito crystallisation robot situated in a cold room at 4 °C. (see Note 12)
    11. The initial hit for the A2AR–mini-Gs414 complex came from the MemGoldTM screen (0.1 M sodium acetate pH 5.5, 8.8% [w/v] PEG 2000 MME). The two crystals that were used for structure determination were grown in either: 0.1 M sodium acetate pH 5.5, 10% (w/v) PEG 2000; or 0.1 M sodium acetate pH 5.7, 9.5% (w/v) PEG 2000 MME.
    12. Harvest the crystals in a cold room at 4 °C. Sequentially transfer the crystals between drops of mother liquor supplemented with increasing concentrations (10, 20, 30%) of the cryoprotectant PEG 400 (1 min in each solution), before flash freezing in liquid nitrogen. (see Note 13)

Data analysis

Chromatograms were visualised using UNICORN software and graphs were plotted using GraphPad Prism 7. Statistical analysis was not required for this work.

Notes

  1. The A2AR construct described here contains a deletion to remove the flexible C-terminus, the length of this deletion was one area of optimisation used to improve the diffraction of the A2AR–mini-Gs crystals. Crystallisation trials of the A2AR–mini-Gs complex were performed using A2AR constructs that consisted of residues 1-308, 1-311 or 1-317. The best diffraction was observed using the construct consisting of residues 1-308 of A2AR. The A2AR–mini-Gs structure (Carpenter et al., 2016) revealed that the C-terminal region of A2AR is involved in crystal contacts, which explains why A2AR constructs of different lengths affected the crystallisation of the complex.
  2. A C-terminal 10x histidine tag and TEV protease cleavage site were utilised because this strategy has previously been used by our lab to purify and crystallise a thermostabilised A2AR construct (Lebon et al., 2011a and 2011b; Lebon and Tate, 2015). A 10x histidine tag is preferred over a 6x histidine tag because it binds Ni2+-NTA resin with higher affinity and therefore allows higher imidazole concentrations (up to 80 mM) to be used during the wash step, which significantly improves the purity of the receptor.
  3. It is important to fill the Ti45 ultracentrifuge tubes correctly (to the neck). Partially filled tubes can collapse during centrifugation causing damage to the cap assembly and rotor.
  4. Filtration of the supernatant usually requires six Steritop filter units due to its high viscosity and the presence of particulate matter. However, this step greatly extends the life of the Ni2+-NTA column, which can be regenerated and reused up to 10 times.
  5. It is important to use a refrigerated centrifuge when concentrating A2AR to maintain the temperature at a constant 4 °C. A non-refrigerated centrifuge located in a cold room will rapidly warm up during operation, which will result in the denaturation of the temperature-sensitive receptor.
  6. Following buffer exchange, and during the overnight incubation with TEV protease, a white precipitate may be observed. The composition of this precipitate is unknown, but it does not contain a significant amount of A2AR and can be removed by centrifugation at 5,000 x g for 5 min at 4 °C prior to the Ni2+-NTA negative purification step.
  7. A2AR usually elutes from the preparative gel filtration column as a broad asymmetric peak of 3-4 ml in volume. The reason for this is unknown, but it may be due to excess detergent, carried over from the solubilisation and purification, affecting the migration of A2AR through the gel filtration matrix. After pooling and concentrating the peak fractions from the preparative gel filtration run, a small analytical sample of A2AR (~100 μg) can be re-run on the same column. This sample should resolve as a sharp peak and is a better indicator of the quality of the purified receptor than the preparative gel filtration profile.
  8. The amido black assay (Schaffner and Weissmann, 1973) is one of the most reliable ways to accurately determine the concentration of membrane proteins in the presence of detergent and ligand.
  9. A2AR that has been frozen and thawed retains its ability to form a complex with mini-Gs and this complex can be crystallised with similar results to those observed when using freshly prepared receptor. Buffer E contains 10% glycerol, so additional cryoprotectants are not required.
  10. The shortest incubation time tested was three hours, after which the A2AR–mini-Gs complex was fully formed. However, an overnight incubation does not negatively affect the stability of the complex and is often more convenient.
  11. Membrane protein samples should never be boiled before loading on SDS-PAGE because this causes the protein to aggregate.
  12. Crystallisation plates must be setup and incubated at 4 °C, even short term exposure to elevated temperatures will result in denaturation of the complex.
  13. Cryoprotection using 30% PEG 400 resulted in visible shrinkage of the crystals. The extent of this dehydration varied between crystals, meaning that datasets collected from different crystals were difficult to merge. Increasing the concentration of the cryoprotectant in a stepwise manner resulted in more uniform dehydration of the crystals, and allowed the collection of diffraction data that could be reliably merged.
  14. PMSF solutions must be prepared in an anhydrous solvent (such as absolute ethanol), because even a small water content will cause rapid hydrolysis of the PMSF rendering it inactive. PMSF stock solutions prepared in absolute ethanol are stable and can be conveniently stored at 4 °C for several months. Note that the PMSF solution should be warmed to room temperature before opening to prevent condensation entering the solvent.

Recipes

  1. Buffer A
    20 mM HEPES, pH 7.5
    1 mM EDTA
    1 mM PMSF (add immediately before use)
  2. PMSF stock solution
    200 mM in absolute ethanol, store at 4 °C (see Note 14)
  3. NECA stock solution
    10 mM in DMSO, store at -20 °C
  4. DM stock solution
    20% w/v in Milli-Q water, store at -20 °C
  5. Buffer B
    20 mM HEPES, pH 7.5
    300 mM NaCl
    10 mM imidazole
    100 μM NECA (add immediately before use)
    0.15% (w/v) DM (add immediately before use)
    Filter using a 0.22 μm Steritop or syringe filter
  6. Buffer C
    20 mM HEPES, pH 7.5
    500 mM NaCl
    80 mM imidazole
    10% (v/v) glycerol
    100 μM NECA (add immediately before use)
    0.15 % (w/v) DM (add immediately before use)
    Filter using a 0.22 μm Steritop or syringe filter
  7. Buffer D
    20 mM HEPES, pH 7.5
    100 mM NaCl
    300 mM imidazole
    10% (v/v) glycerol
    100 μM NECA (add immediately before use)
    0.15% (w/v) DM (add immediately before use)
    Filter using a 0.22 μm Steritop or syringe filter
  8. Buffer E
    10 mM HEPES, pH 7.5
    100 mM NaCl
    10% (v/v) glycerol
    100 μM NECA (add immediately before use)
    0.15% (w/v) DM (add immediately before use)
    Filter using a 0.22 μm Steritop or syringe filter
  9. OTG stock solution
    15% w/v in Milli-Q water, store at -20 °C
  10. Apyrase stock solution
    1 unit/µl in Milli-Q water, store at -20 °C
  11. Buffer F
    10 mM HEPES, pH 7.5
    100 mM NaCl
    1 mM MgCl2
    100 μM NECA (add immediately before use)
    0.35% (w/v) OTG (add immediately before use)
    Filter using a 0.22 μm Steritop or syringe filter

Acknowledgments

This work was funded by a grant from Heptares Therapeutics Ltd and core funding from the Medical Research Council [MRC U105197215]. Parts of this protocol was adapted from previously described methods (Lebon et al., 2011a and 2011b; Tate and Lebon, 2015; Carpenter et al., 2016). We thank Rony Nehmé for comments on the manuscript.

References

  1. Carpenter, B., Nehmé, R., Warne, T., Leslie, A. G. and Tate, C. G. (2016). Structure of the adenosine A2A receptor bound to an engineered G protein. Nature 536(7614): 104-7.
  2. Carpenter, B. and Tate, C. G. (2016). Engineering a minimal G protein to facilitate crystallisation of G protein-coupled receptors in their active conformation. Protein Eng Des Sel 29(12): 583-594.
  3. Carpenter, B. and Tate, C. G. (2017). Expression and purification of mini G proteins from Escherichia coli. Bio-protocol 7(8): e2235.
  4. Lebon, G., Bennett, K., Jazayeri, A. and Tate, C. G. (2011a). Thermostabilisation of an agonist-bound conformation of the human adenosine A2A receptor. J Mol Biol 409(3): 298-310.
  5. Lebon, G., Warne, T., Edwards, P. C., Bennett, K., Langmead, C. J., Leslie, A. G. and Tate, C. G. (2011b). Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation. Nature 474: 521-5.
  6. Schaffner, W. and Weissmann, C. (1973). A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem 56(2): 502-514.
  7. Tate, C. G. and Lebon, G. (2015). Purification and crystallization of a thermostabilized agonist-bound conformation of the human adenosine A2A receptor. Methods Mol Biol 1335: 17-27.

简介

G蛋白偶联受体(GPCR)通过激活异源三聚体G蛋白来响应细胞外刺激如光,激素和核苷来促进细胞质信号传导。 GPCR-G蛋白复合物的结构测定对于了解信号转导的精确机制至关重要。然而,由于它们的构象动态和固有的短暂性质,这些复合物是结构研究的具有挑战性的目标。我们最近开发了一种工程化的G蛋白,微型G ,解决了这些问题,并允许形成稳定的GPCR-G蛋白复合物。 Mini-G 促进了人腺苷A 2A受体(A 2A 2A)在其G蛋白结合构象中的结构测定,在3.4 Å分辨率。在这里,我们描述了A 2A R R的表达和纯化的一步一步的方案,并且A 2AA-R-mini-G'子>复杂。

背景 我们最近开发了一种工程化的最小G蛋白,迷你G(Carpenter和Tate,2016),其促进了人腺苷A 2A受体的结构测定(A <其活性状态(Carpenter等人,2016)。 Mini-G 充分稳定A 2A R的活性构象,以允许络合物在洗涤剂辛硫基葡糖苷中通过蒸气扩散结晶。在这里,我们描述了一种用于表达和纯化Aβ2A R的详细方案,该方案根据我们实验室开发的先前描述的方法(Lebon等人,2011a和2011b;泰特和Lebon,2015)。我们还描述了先前在Carpenter等人的描述中的A 2A'R-mini-G'复合物的制备和结晶的一步一步的方法, (2016)。在同伴手稿(Carpenter和Tate,2017)中描述了迷你G 的表达和纯化。

关键字:腺苷A2A受体, A2AR, 活性状态, GPCR, G蛋白偶联受体, 迷你G蛋白, 迷你Gs, G蛋白复合物

材料和试剂

  1. 血清移液管
  2. 移液器提示(STARLAB INTERNATION)
  3. 塑料铲
  4. 50ml管(SARSTEDT,目录号:62.547.254)
  5. 15 ml管(SARSTEDT,目录号:62.554.002)
  6. (Eppendorf,目录号:0030119401)
  7. 1.5毫升管(SARSTEDT,目录号:72.690.001)
  8. 0.5ml管(SARSTEDT,目录号:72.699)
  9. Steritop0.22μm过滤器(EMD Millipore,目录号:SCGPT01RE)
  10. Amicon Ultra-15集中器50 kDa截止(EMD Millipore,目录号:UFC905024)
  11. 塑料柱(例如,空的PD-10柱)(GE Healthcare,目录号:17043501)
  12. PD-10脱盐柱(GE Healthcare,目录号:17085101)
  13. Amicon Ultra-4集中器50 kDa截止(EMD Millipore,目录号:UFC805024)
  14. MRC 96孔2滴结晶板(分子尺寸,目录号:MD11-00-100)
  15. Ni 2 + -NTA超流5ml预填充柱(QIAGEN,目录号:30760)
  16. Superdex 200 GL 10/300凝胶过滤柱(GE Healthcare,目录号:17517501)
  17. 昆虫细胞系(表达系统,目录号:94-002F)
    Trichoplusia ni
  18. pBacPAK8质粒(Takara Bio,Clontech,目录号:PT1262-5)
  19. flashBAC ULTRA DNA(Oxford Expression Technologies,目录号:100300)
  20. ESF921昆虫细胞培养基(Expression Systems,目录号:96-001-01)
  21. 胎牛血清(Sigma-Aldrich,目录号:F9665)
  22. cOmplete,无EDTA的蛋白酶抑制剂片剂(Roche Diagnostics,目录号:11873580001)
  23. PMSF(Sigma-Aldrich,目录号:P7626)
  24. 液氮
  25. NECA(Sigma-Aldrich,目录号:E2387)
  26. 咪唑(Sigma-Aldrich,目录号:56748)
  27. 氯化钠(NaCl)(Fisher Scientific,目录号:10598630)
  28. N-癸基β-麦芽糖苷(DM)洗涤剂(Anatrace,目录号:D322)
  29. TEV蛋白酶(内部生产)
  30. Ni 2 + -NTA琼脂糖(QIAGEN,目录号:30210)
  31. 氯化镁(MgCl 2)(Fisher Scientific,目录号:BP214-500)
  32. Apyrase(Sigma-Aldrich,目录号:A6535)
  33. 乙酸钠(Fisher Scientific,目录号:10794761)
  34. MemGold TM 结晶屏幕(分子尺寸,目录号:MD1-39)
  35. PEG 2000(Sigma-Aldrich,目录号:81221)
    注意:本产品已停产。
  36. PEG 2000 MME(Sigma-Aldrich,目录号:81321)
    注意:本产品已停产。
  37. PEG 400(Hampton Research,目录号:HR2-603)
  38. 甘油(VWR,目录号:24388.320)
  39. HEPES(Sigma-Aldrich,目录号:H3375)
  40. EDTA
  41. 绝对乙醇(VWR,目录号:20821.330)
  42. DMSO(Sigma-Aldrich,目录号:D2650)
  43. 正辛基-β-D-硫代葡糖苷(OTG)洗涤剂(Glycon,目录号:D20014)
  44. Precision Plus SDS-PAGE分子量标准(Bio-Rad Laboratories,目录号:161-0373)
  45. 4-20%Tris-甘氨酸SDS-PAGE凝胶(Fisher Scientific,目录号:EC60255BOX)
  46. 缓冲液A(参见食谱)
  47. PMSF储备溶液(见配方)
  48. NECA库存解决方案(见配方)
  49. DM储备溶液(参见食谱)
  50. 缓冲液B(参见食谱)
  51. 缓冲区C(见配方)
  52. 缓冲区D(见配方)
  53. 缓冲液E(参见食谱)
  54. OTG储备溶液(见配方)
  55. Apyrase储备溶液(见配方)
  56. 缓冲区F(见配方)

设备

  1. 移液器(STARLAB INTERNATION)
  2. 最佳生长 TM 5L烧瓶(Thompson Instrument Company,目录号:931116)
  3. Shaker孵化器(Infors,型号:Multitron Standard)
  4. 高速离心机(例如,Beckman Coulter,型号:Avanti J-26XP,目录号:393124)
  5. 45型Ti(Ti45)超速离心机转子(Beckman Coulter,目录号:339160)
  6. 45型Ti(Ti45)超离心瓶组件(Beckman Coulter,目录号:355622)
  7. 水浴(Julabo)
  8. ULTRA-TURRAX T25均质机(IKA,型号:ULTRA-TURRAX T25均质器)
  9. 磁力搅拌棒
  10. 70型Ti(Ti70)超离心转子(Beckman Coulter,目录号:337922)
  11. 70型Ti(Ti70)超离心瓶组件(Beckman Coulter,目录号:355618)
  12. 蠕动泵(例如,GE Healthcare,型号:Pump P-1,目录号:18-1110-91)
  13. 冷藏式台式离心机(例如,Eppendorf,目录号:5430R)
  14. 辊式搅拌机(IKA,型号:ROLLER 6数码,目录号:0004011000)
  15. TLA55台式超速离心机转子(Beckman Coulter,目录号:366725)
  16. Optima L100 XP制备型超速离心机(Beckman Coulter,型号:Optima TM L-100 XP)
  17. Optima MAX台式超速离心机(Beckman Coulter,型号:Optima TM MAX)
  18. 冷冻微量离心机(例如,Eppendorf,目录号:5418R)
  19. 转子能够旋转1升瓶(例如,JLA-8.1000)(Beckman Coulter,目录号:363688)
  20. ÄKTA净化器色谱系统(GE Healthcare,型号:ÄKTA净化器)
  21. Mosquito ®晶体蛋白结晶机器人(TTP Labtech,型号:mosquito Crystal)

软件

  1. UNICORN(GE Healthcare)
  2. 图形软件(例如,,Prism 7)(GraphPad)

程序

  1. A 2A细胞在昆虫细胞中的表达
    截短以去除柔性C末端(见注1)并含有N154A突变以除去潜在的N末端糖基化位点的人A 2A'R是克隆到转移载体pBacPAK8中,并按照制造商的说明书使用flashBAC ULTRA系统制备杆状病毒。 A 2A R构建体含有C末端10x组氨酸标签以促进纯化(参见附注2)和TEV蛋白酶切割位点以允许去除组氨酸标签(图1)。


    图1:2A亚型R杆状病毒表达构建体。使用Bam 将2A亚基R克隆到杆状病毒转移载体pBacPAK8中> HI和不是 I限制性位点(下划线),并且使用flashBAC ULTRA系统制备杆状病毒。该构建体由人A 2A亚基残基1-308,然后是TEV蛋白酶切割位点(以绿色突出显示)和C末端10x组氨酸标签(以红色突出显示)组成。 2A R含有N154A突变(以黄色突出显示)以除去潜在的N末端糖基化位点。开始和结束密码以粗体显示。

    1. 稀释T (在ESF921无血清培养基中生长)以5×10 6细胞/ml的密度在5L最佳生长烧瓶中加入2,730ml,并在27℃下孵育过夜C,以124rpm振荡
    2. 一旦密度达到3.3×10 6细胞/ml,加入150ml胎牛血清(终浓度为5%v/v)和120ml第3代A 2A R杆状病毒(终浓度为4%v/v)
    3. 在27℃孵育65-72小时,以124rpm摇动。
    4. 通过在4℃下以5,000×g离心10分钟来收获细胞。
    5. 将细胞沉淀重悬于缓冲液A中的最终体积为180ml,加入4种蛋白酶抑制剂片剂和PMSF,得到终浓度为1mM。闪光在液氮中冷冻并储存在-80°C
  2. 昆虫细胞膜制备
    进行粗制膜,其保留所有不溶性细胞内成分,同时除去可溶性蛋白质。这种类型的膜制备有助于限制膜片段的损失并最大化2A 2A的回收。该方案描述了来自三升昆虫细胞培养物的膜制备,其对于一个2AR2R纯化是足够的。然而,如果需要,可以使用单个Ti45超速离心机转子同时处理两批(总共六升培养物)。
    1. 在室温下在水浴中从3L昆虫细胞培养物中解冻细胞沉淀
    2. 添加新鲜的PMSF以使终浓度为1mM,将溶液转移到三个Ti45管中,并在Ti45转子中以158,000×g(45,000rpm)在4℃下离心2小时。确保管子正确填充(颈部),如有必要,补充缓冲液A. (见注3)
    3. 小心地除去上清液,直到与血清学移液管的颗粒顶部水平一致(见图2)

      图2.超离心后膜片的图示超速离心后,膜片颗粒的表面上观察到含有大量A 2A R的扩散层。重要的是不要倾析上清液,因为这将去除扩散层,导致A 2A R R的产率降低。相反,应使用血清移液管小心吸取上清液,直至与膜片的顶部水平(用虚线表示)。

    4. 向每个管中加入25毫升缓冲液A,并使用塑料刮刀将颗粒破碎成小块
    5. 使用以10,000rpm运行的ULTRA-TURRAX T25均质器均质沉淀,直至不残留块状物。每管约需30秒。
    6. 用缓冲液A加热管,并在4℃下在Ti45转子中以158,000 x g(45,000rpm)离心2小时。 (见注3)
    7. 用血清移液管取出上清液直至与沉淀物顶部平齐(见图2)
    8. 使用塑料刮刀,然后用ULTRA-TURRAX T25匀浆器将剩余的缓冲液重悬于管中。不要超过160毫升的终体积。将膜悬浮液等分成50ml管,在液氮中闪蒸冷冻,并储存在-80℃。膜可以在这些条件下储存长达三个月。

  3. A 2A R的纯化 在激动剂NECA的存在下,在癸基麦芽糖苷(DM)中纯化A 2A'R,使用从先前描述的用于纯化热稳定的A 2AA的方法, R建筑(Lebon等人,2011a和2011b; Tate和Lebon,2015)。如果将复合物稍后更换成用于蒸气扩散结晶的短链洗涤剂,则必须使用DM。该方案通常产生3-6mg来自3L昆虫细胞培养物的纯A 2AA R。
    1. 在室温下在水浴中从3L昆虫细胞培养物中解冻膜。
    2. 加入4种蛋白酶抑制剂片剂,NECA(100μM),咪唑(10mM),PMSF(1mM)和NaCl(300mM),得到最终浓度,室温搅拌30分钟,以允许激动剂结合。
    3. 将膜悬浮液在冰浴中冷却至4℃。使用磁力搅拌棒快速搅拌,通过加入DM来提供最终浓度为2%(w/v)的膜,使其在4℃下缓慢搅拌30分钟。
    4. 将溶液转移到8个Ti70管中,并在4℃下在Ti70转子中以265,000 x g(60,000 rpm)离心2小时。
    5. 取出上清液并通过0.2μmSteritop过滤器过滤。 (见注4)
    6. 在4℃下以2ml/min的流速将滤液(〜150ml)装入5ml Ni 2 O 3 -NTA Superflow柱(用缓冲液B预平衡)中,使用蠕动泵。滤液在装载时应保持在冰上。
    7. 用100ml缓冲液C以4ml流速5ml/min洗涤柱子。
    8. 在4℃以2ml/min的流速用20ml缓冲液D洗脱柱,收集单一的20ml级分。
    9. 在4℃的冷藏台式离心机中,使用Amicon Ultra-15浓缩器(50kDa分子量截留)将洗脱液浓缩至5ml。 (见注5)
    10. 使用两个PD-10脱盐柱(每个柱上加入2.5ml)将浓缩的蛋白质交换到缓冲液E中。 (见注6)
    11. 加入TEV蛋白酶,得到约1:3(w/w)的TEV:A 2AR比,在冰上孵育过夜。
    12. 加入2ml Ni-sup + -NTA琼脂糖树脂(用缓冲液E预平衡),在辊式混合器中在4℃下混合30分钟。
    13. 将悬浮液倒入1ml装在一次性塑料柱中的Ni 2+/NTA琼脂糖树脂(用缓冲液E预平衡),使其通过重力流过。
    14. 收集流过并用2×3ml缓冲液E洗涤柱。
    15. 在4℃的冷藏台式离心机中使用Amicon Ultra-15浓缩器(50kDa截留)浓缩至0.2ml浓缩至0.2ml。
    16. 在TLA55转子中使用台式超速离心机在4℃下离心135,000 x g(55,000 rpm)的浓缩蛋白质10分钟以去除聚集体。
    17. 将上清液加载到Superdex 200 GL 10/300凝胶过滤柱(用缓冲液E预平衡)中,在4℃以0.4ml/min的流速,收集0.5ml级分。
    18. 基于凝胶过滤色谱的池峰分数(通常为3-4ml)。 (见注7)
    19. 使用Amicon Ultra-4浓缩器(50 kDa截止值)在4°C的冷藏台式离心机中将合并的馏分浓缩至0.2 ml。
    20. 使用酰胺黑测定法确定蛋白质浓度(Schaffner和Weissmann,1973)。 (见注8)
    21. 如果需要,纯化的A 2A R可以等分,在液氮中快速冷冻并储存在-80℃。 (见注9) 

  4. A 2A< R-mini-G>>复合物的结晶
    首先在DM中制备A 2A'R-mini-G'复合物,一旦形成复合物,其稳定足够被交换成用于蒸气扩散的辛基硫代葡萄糖苷(OTG)结晶试验。在同伴手稿(Carpenter and Tate,2017)中提供了用于表达和纯化mini-G 的详细方案。
    1. 将2mg A 2AA R等分到微量离心管中并加入1.2倍摩尔过量的mini-G ++构建体414(1.8mg)。用缓冲液E将体积调节至0.2ml,加入MgCl 2,终浓度为1mM,加入0.1单位的apyrase。在冰上孵育3-16小时。 (见注10)
    2. 通过稀释缓冲液F中的复合物1:10开始洗涤剂交换(从DM到OTG),并使用Amicon Ultra-4浓缩器(50kDa截止值)在4°冷藏台式离心机中再浓缩至0.2ml C.
    3. 使用台式超速离心机在4°C下离心TLA55转子55,000 rpm(135,000 x g)的浓缩蛋白,以清除聚集体。
    4. 通过将上清液加载到Superdex 200 GL 10/300凝胶过滤柱(用缓冲液F预平衡)中,在4℃以0.5ml/min的流速加载洗涤剂交换,收集0.5ml级分。
    5. 基于凝胶过滤色谱图的池峰分数(通常为2-3ml)(图3)

      图3.A.2A R R纯化的SDS-PAGE和凝胶过滤分析.A和B。代表性A 2A的纯化的SDS-PAGE分析 R构造。尽管分子量分别为35和27kDa,A 2 R R和mini-G 4 414在SDS-PAGE凝胶上以相同的速率迁移,使得可视化复杂难因此,为了说明的目的,我们显示了包含N-末端硫氧还蛋白融合物(TrxA-A 2AiR)的A 2AA构建体的SDS-PAGE凝胶,其增加其分子量至46kDa。使用与对A 2A R所述相同的方案,在DM中纯化TrxA-A 2AA,然而TrxA-A 2A-R-被交换成OTG的mini-G 复合物没有产生衍射到高于分辨率的晶体。 A.TrxA-A 2A纯化的SDS-PAGE分析:(M)分子量标记; (1)Ni 2 + -NTA柱装载材料(1:10稀释); (2)Ni 2 + -NTA柱流过(1:10稀释); (3)Ni 2 + -NTA柱洗; (4)Ni 2 + -NTA柱洗脱液; (5)浓缩和缓冲液交换后的样品; (6)TEV消化后样品; (7)Ni 2 + -NTA负净化流过; (8)Ni 2 + -NTA阴性纯化洗脱液; (9)凝胶过滤池。 TEV蛋白酶由星号表示。在加入凝胶之前,不应将样品煮沸(见附注11)。 B.洗涤剂交换成OTG:(M)分子量标记物后,TrxA-A 2A R-mini-G 404复合物的SDS-PAGE分析; (1)凝胶过滤池。 C.在OTG中的A2A-R-mini-G 404复合物的合成凝胶过滤色谱图,合并的级分(通常为2-3ml)由虚线表示。请注意,紫外检测器达到饱和,这导致截断的吸光度分布。 D.在欧洲同步加速器辐射设备(尺度标尺为...)处安装在束线ID23-2上的环路中的A 2A'R-mini-G 404复合物的晶体50 x 100μm)
    6. 使用Amicon Ultra-4浓缩器(50kDa截留)将浓缩的馏分浓缩至≥20mg/ml,在4℃的冷冻台式离心机中。
    7. 在TLA55转子中使用台式超速离心机在4℃下离心135,000 x g(55,000 rpm)的浓缩蛋白质10分钟以去除聚集体。
    8. 将上清液转移到新管中,弃去沉淀。
    9. 使用酰胺黑测定准确测定复合物的浓度(Schaffner和Weissmann,1973)
    10. 将复合物稀释至20 mg/ml,并立即在96孔坐落液滴板中设置结晶试验,使用位于4°C的冷室中的蚊子结晶机器人,分配120 n1蛋白质和120 nl沉淀剂溶液。 (见注12)
    11. A 2A> R-mini-G 404复合体的初始命中来自MemGold< sup>< sup>筛选(0.1M醋酸钠pH5.5, 8.8%[w/v] PEG 2000 MME)。用于结构测定的两种晶体生长在:0.1M乙酸钠pH 5.5,10%(w/v)PEG 2000中;或0.1M乙酸钠pH 5.7,9.5%(w/v)PEG 2000 MME。
    12. 在4°C的寒冷室中收获晶体。在液氮中快速冷冻之前,将补充有浓度增加浓度(10%,20%,30%)冷冻保护剂PEG 400(每种溶液1分钟)的母液滴转移到晶体上。 (见附注13)

数据分析

使用UNICORN软件可视化色谱图,并使用GraphPad Prism 7绘制图形。本工作不需要统计学分析。

笔记

  1. 这里描述的A 2A R构型包含去除柔性C-末端的缺失,该缺失的长度是用于改善A2AR-mini-Gs晶体的衍射的一个优化领域。使用由残基1-308,1组成的A 2A Sub构建体进行A 2AA-R-mini-G n复合物的结晶试验-311或1-317。使用由A 2A R的残基1-308组成的构建体观察到最佳的衍射。 A< 2A> R-mini-G>结构(Carpenter等人,2016)揭示了A 2A R涉及晶体接触,这就解释了为什么不同长度的A 2A R构型影响复合物的结晶。
  2. 使用C末端10x组氨酸标签和TEV蛋白酶切割位点,因为我们的实验室先前已经使用该策略来纯化和结晶热稳定的A 2A/R构建体(Lebon等人, ,2011a和2011b; Lebon和Tate,2015)。 10x组氨酸标签优于6x组氨酸标签,因为它以更高的亲和力结合Ni 2+/NTA树脂,因此允许在洗涤步骤期间使用较高的咪唑浓度(高达80mM)这显着提高了受体的纯度
  3. 重要的是要正确填充Ti45超速离心管(颈部)。部分填充的管可能在离心过程中塌陷,从而损坏盖组件和转子
  4. 由于其高粘度和颗粒物质的存在,上清液的过滤通常需要六个Steritop过滤器单元。然而,这一步大大延长了Ni 2 + -NTA色谱柱的使用寿命,可以再生10次。
  5. 当浓缩A 2A R以使温度保持在4℃时,重要的是使用冷藏式离心机。位于寒冷室内的非冷冻离心机将在运行过程中迅速升温,这将导致温度敏感受体的变性。
  6. 缓冲液交换后,在与TEV蛋白酶过夜温育期间,可观察到白色沉淀。该沉淀物的组成是未知的,但它不含有显着量的A2AR,并且可以在4℃下以5,000×g离心5分钟除去Ni 2+ -NTA负净化步骤。
  7. A 2A R通常从制备型凝胶过滤柱洗脱,为体积为3-4ml的宽不对称峰。其原因尚不清楚,但可能是由于增溶和净化所带来的过量洗涤剂,会影响Aβ2A R通过凝胶过滤基质的迁移。在制备凝胶过滤运行中汇集并浓缩峰分数后,可以在同一柱上重新运行A 2A R(〜100μg)的小分析样品。该样品应该作为尖峰解析,并且比制备型凝胶过滤曲线更好地指示纯化受体的质量。
  8. 酰胺黑测定(Schaffner和Weissmann,1973)是在洗涤剂和配体存在下准确测定膜蛋白浓度的最可靠方法之一。
  9. 已经冷冻和解冻的2A 2A保留其形成具有小-Gas的复合物的能力,并且该复合物可以结晶,与使用新鲜时观察到的结果相似。制备受体。缓冲液E含有10%甘油,因此不需要额外的冷冻保护剂。
  10. 测试的最短孵育时间为3小时,之后完全形成A 2A'R-mini-G'复合物。然而,过夜孵化不会对复合物的稳定性产生负面影响,而且通常更为方便
  11. 膜蛋白样品在加载SDS-PAGE之前不应该煮沸,因为这会使蛋白质聚集
  12. 结晶板必须设置并在4℃下孵育,即使短期暴露于高温也会导致复合物变性。
  13. 使用30%PEG 400的低温保护导致晶体的可见收缩。这种脱水的程度在晶体之间变化,意味着从不同晶体收集的数据集难以合并。逐步增加冷冻保护剂的浓度导致晶体更均匀的脱水,并允许收集可靠可靠的衍射数据。
  14. PMSF溶液必须在无水溶剂(如无水乙醇)中制备,因为即使少量的水分会导致PMSF的快速水解,使其无活性。以无水乙醇制备的PMSF储备溶液是稳定的,可以方便地在4℃下储存几个月。注意,PMSF溶液在打开前应加热至室温,以防止冷凝物进入溶剂。

食谱

  1. 缓冲区A
    20 mM HEPES,pH 7.5
    1 mM EDTA
    1毫升PMSF(立即使用前添加)
  2. PMSF库存解决方案
    无水乙醇200mM,储存于4℃(见附注14)
  3. NECA库存解决方案
    在DMSO中10 mM,储存于-20°C
  4. DM股票解决方案
    在Milli-Q水中20%w/v,储存于-20°C
  5. 缓冲区B
    20 mM HEPES,pH 7.5
    300 mM NaCl
    10 mM咪唑
    100μMNECA(使用前立即添加)
    0.15%(w/v)DM(立即使用前添加)
    使用0.22微米的Steritop或注射器过滤器过滤
  6. 缓冲区C
    20 mM HEPES,pH 7.5
    500 mM NaCl
    80 mM咪唑
    10%(v/v)甘油 100μMNECA(使用前立即添加)
    0.15%(w/v)DM(立即使用前添加)
    使用0.22微米的Steritop或注射器过滤器过滤
  7. 缓冲区D
    20 mM HEPES,pH 7.5
    100 mM NaCl
    300毫克咪唑
    10%(v/v)甘油 100μMNECA(使用前立即添加)
    0.15%(w/v)DM(立即使用前添加)
    使用0.22微米的Steritop或注射器过滤器过滤
  8. 缓冲区E
    10mM HEPES,pH7.5
    100 mM NaCl
    10%(v/v)甘油 100μMNECA(使用前立即添加)
    0.15%(w/v)DM(立即使用前添加)
    使用0.22微米的Steritop或注射器过滤器过滤
  9. OTG库存解决方案
    在Milli-Q水中为15%w/v,储存于-20°C
  10. Apyrase储备溶液
    在Milli-Q水中1单位/μl,储存于-20°C
  11. 缓冲区F
    10mM HEPES,pH7.5
    100 mM NaCl
    1mM MgCl 2
    100μMNECA(使用前立即添加)
    0.35%(w/v)OTG(立即使用前添加)
    使用0.22微米的Steritop或注射器过滤器过滤

致谢

这项工作由Heptares Therapeutics有限公司的资助和医学研究委员会的核心资金[MRC U105197215]资助。该协议的一部分是从先前描述的方法(Lebon等人,2011a和2011b; Tate和Lebon,2015; Carpenter等人,2016)改编的。我们感谢RonyNehmé对手稿的意见。

参考文献

  1. Carpenter,B.,Nehmé,R.,Warne,T.,Leslie,AG和Tate,CG(2016)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm。 nih.gov/pubmed/27462812"target ="_ blank">与工程化G蛋白结合的腺苷A 2A亚型的结构自然 536(7614 ):104-7。
  2. Carpenter,B.和Tate,CG(2016)。工程化最小的G蛋白质以促进G蛋白偶联受体在其活性构象中的结晶。蛋白质工程技术29(12):583-594。
  3. Carpenter,B. and Tate,CG(2017)。  表达和纯化大肠杆菌中的迷你G蛋白质。生物方案7(8):e2235。
  4. Lebon,G.,Bennett,K.,Jazayeri,A.和Tate,CG(2011a)。  人腺苷A 2A受体的激动剂结合构象的热稳定化.Mol Biol 409(3) :298-310。
  5. Lebon,G.,Warne,T.,Edwards,PC,Bennett,K.,Langmead,CJ,Leslie,AG and Tate,CG(2011b)。< a class ="ke-insertfile"href ="https: //www.ncbi.nlm.nih.gov/pubmed/21593763"target ="_ blank">激动剂结合的腺苷A 2A亚型受体结构揭示了GPCR激活的常见特征。 >自然 474:521-5。
  6. Schaffner,W.和Weissmann,C.(1973)。用于测定稀释溶液中蛋白质的快速,灵敏和具体的方法。 Anal Biochem 56(2):502-514。
  7. Tate,CG和Lebon,G.(2015)。  人腺苷A 2A受体的热稳定激动剂结合构象的纯化和结晶。方法Mol Biol 1335:17-27。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Carpenter, B. and Tate, C. G. (2017). Expression, Purification and Crystallisation of the Adenosine A2A Receptor Bound to an Engineered Mini G Protein. Bio-protocol 7(8): e2234. DOI: 10.21769/BioProtoc.2234.
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