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Expression and Purification of Mini G Proteins from Escherichia coli

迷你G蛋白在大肠埃希杆菌的表达和纯化

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.2235 浏览次数: 13399

评审: Arsalan DaudiShyam SolankiAnonymous reviewer(s)

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Abstract

Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and functional characterisation of GPCR–G protein complexes is important to fully decipher the mechanism of signal transduction. However, native G proteins are unstable and conformationally dynamic when coupled to a receptor. We therefore developed an engineered minimal G protein, mini-Gs, which formed a stable complex with GPCRs, and facilitated the crystallisation and structure determination of the human adenosine A2A receptor (A2AR) in its active conformation. Mini G proteins are potentially useful tools in a variety of applications, including characterising GPCR pharmacology, binding affinity and kinetic experiments, agonist drug discovery, and structure determination of GPCR–G protein complexes. Here, we describe a detailed protocol for the expression and purification of mini-Gs.

Keywords: Complex (复合物) search Engineered G protein (改造的G蛋白) search G protein-coupled receptor (G蛋白偶联受体) search GPCR (GPCR) search Mini G protein (迷你G蛋白) search Mini-Gs (迷你Gs) search

Background

We recently reported the development of an engineered minimal G protein, mini-Gs (Carpenter and Tate, 2016), which facilitated the crystallisation of the human adenosine A2A receptor (A2AR) in its active conformation (Carpenter et al., 2016; Carpenter and Tate, 2017). Unlike heterotrimeric G proteins, which require expression in eukaryotic systems, mini-Gs is highly expressed in Escherichia coli (E. coli) and can be easily purified with a yield of 50-100 mg of mini-Gs per liter of culture. Here, we describe a step by step protocol, earlier described in Carpenter and Tate (2016), that can be used for the expression and purification of any of the mini G protein constructs described previously (Carpenter et al., 2016; Carpenter and Tate, 2016). Since mini-Gs construct 393 is well suited to most applications (see Carpenter and Tate, 2016), it will be used as an example herein.

Materials and Reagents

  1. Steritop 0.22 μm filter unit (EMD Millipore, catalog number: SCGPT01RE )
  2. 50 ml tubes (SARSTEDT, catalog number: 62.547.254 )
  3. 15 ml tubes (SARSTEDT, catalog number: 62.554.002 )
  4. 2 ml tubes (Eppendorf, catalog number: 0030120094 )
  5. 1.5 ml tubes (SARSTEDT, catalog number: 72.690.001 )
  6. 0.5 ml tubes (SARSTEDT, catalog number: 72.699 )
  7. Pipette tips (STARLAB INTERNATIONAL)
  8. Plastic column (e.g., empty PD-10 column) (GE Healthcare, catalog number: 17043501 )
  9. HisTrap Fast Flow 5 ml prepacked columns (GE Healthcare, catalog number: 17-5255-01 )
  10. Amicon Ultra-15 concentrator 10 kDa cut-off (EMD Millipore, catalog number: UFC901024 )
  11. HiLoad 26/600 Superdex 200 PG gel filtration column (GE Healthcare, catalog number: 28989336 )
  12. SnakeSkin Dialysis Tubing 10 kDa cut-off (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 68100 )
  13. E. coli strain BL21-CodonPlus(DE3)-RIL (Agilent Technologies, catalog number: 230245 )
  14. pET15b plasmid (EMD Millipore, catalog number: 69661 )
  15. Ampicillin (Melford Laboratories, catalog number: A0104 )
  16. Chloramphenicol (MP Biomedicals, catalog number: 0219032125 )
  17. Glucose (Formedium, catalog number: GLU03 )
  18. Magnesium sulfate heptahydrate (MgSO4·7H2O) (VWR, catalog number: 25165.260 )
  19. IPTG (Melford Laboratories, catalog number: MB1008 )
  20. Liquid nitrogen
  21. TEV protease (produced in-house)
  22. cOmplete, EDTA-free protease inhibitor tablets (Roche Diagnostics, catalog number: 11873580001 )
  23. Lysozyme (Sigma-Aldrich, catalog number: L6876 )
  24. Imidazole (Sigma-Aldrich, catalog number: 56748 )
  25. Ni2+-NTA agarose (QIAGEN, catalog number: 30210 )
  26. Gel filtration marker kit (Sigma-Aldrich, catalog number: MWGF200 )
  27. TCEP (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 77720 )
  28. Tryptone (Melford Laboratories, catalog number: GT1332 )
  29. Yeast extract (Melford Laboratories, catalog number: GY1333 )
  30. Sodium chloride (NaCl) (Fisher Scientific, catalog number: 10598630 )
  31. Agar
  32. Terrific Broth (TB) (Melford Laboratories, catalog number: GT1702 )
  33. Glycerol (VWR, catalog number: 24388.320 )
  34. PMSF (Sigma-Aldrich, catalog number: P7626 )
  35. Absolute ethanol (VWR, catalog number: 20821.330 )
  36. Pepstatin-A (Sigma-Aldrich, catalog number: P4265 )
  37. DMSO (Sigma-Aldrich, catalog number: D2650 )
  38. Leupeptin (Sigma-Aldrich, catalog number: L2884 )
  39. DNase I (Sigma-Aldrich, catalog number: DN25 )
  40. DTT (Melford Laboratories, catalog number: MB1015 )
  41. GDP (Sigma-Aldrich, catalog number: G7127 )
  42. HEPES (Sigma-Aldrich, catalog number: H3375 )
  43. Magnesium chloride (MgCl2) (Fisher Scientific, catalog number: BP214-500 )
  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. TYE agar plates (see Recipes)
  47. Luria Bertani (LB) media (see Recipes)
  48. Terrific Broth (TB) media (see Recipes)
  49. PMSF stock solution (see Recipes)
  50. Pepstatin-A stock solution (see Recipes)
  51. Leupeptin stock solution (see Recipes)
  52. DNase I stock solution (see Recipes)
  53. Lysozyme stock solution (see Recipes)
  54. DTT stock solution (see Recipes)
  55. GDP stock solution (see Recipes)
  56. Buffer A (see Recipes)
  57. Buffer B (see Recipes)
  58. Buffer C (see Recipes)
  59. Buffer D (see Recipes)
  60. Buffer E (see Recipes)

Equipment

  1. 2 L Erlenmeyer flasks (e.g., Corning, catalog number: 4980-2L )
  2. High speed centrifuge (e.g., Beckman Coulter, model: Avanti J-26XP , catalog number: 393124)
  3. Magnetic stirring bar
  4. Shaker incubator (Infors, model: Multitron Standard )
  5. Pipettes (STARLAB INTERNATIONAL)
  6. Sonicator equipped with 13 mm probe (e.g., Sonics Vibra-Cell) (Sonics, model: VCX 130 )
  7. Rotor capable of spinning 250 ml bottles (e.g., Beckman Coulter JLA-16.250) (Beckman Coulter, catalog number: 363934 )
  8. Peristaltic pump (e.g., GE Healthcare, model: Pump P-1, catalog number: 18-1110-91 )
  9. NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )
  10. UV/VIS spectrophotometer
  11. Roller mixer (IKA, model: ROLLER 6 digital , catalog number: 0004011000)
  12. Rotor capable of spinning 1 L bottles (e.g., Beckman Coulter JLA-8.1000) (Beckman Coulter, catalog number: 363688 )
  13. Refrigerated benchtop centrifuge (e.g., Eppendorf, catalog number: 5430 R )
  14. Refrigerated microcentrifuge (e.g., Eppendorf, catalog number: 5418 R )
  15. ÄKTA Purifier chromatography system (GE Healthcare, model: ÄKTA Purifier )

Software

  1. UNICORN (GE Healthcare)
  2. Graphical software (e.g., Prism 7) (GraphPad), or free alternatives (e.g., R Bioconductor packages) (Bioconductor)

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 and Purification of Mini G Proteins from Escherichia coli. Bio-protocol 7(8): e2235. DOI: 10.21769/BioProtoc.2235.

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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 > 表达

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