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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

Byron Carpenter Byron Carpenter
Christopher G. Tate Christopher G. Tate

发布: 2017年04月20日第7卷第8期 DOI: 10.21769/BioProtoc.2234 浏览次数: 12200

评审: Arsalan DaudiAdam IdoineYoko Eguchi

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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) search A2AR (A2AR) search Active state (活性状态) search GPCR (GPCR) search G protein-coupled receptor (G蛋白偶联受体) search Mini G protein (迷你G蛋白) search Mini-Gs (迷你Gs) search G protein complex (G蛋白复合物) search

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

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

生物化学 > 蛋白质 > 分离和纯化

生物化学 > 蛋白质 > 结构

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