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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   

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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), Engineered G protein(改造的G蛋白), G protein-coupled receptor(G蛋白偶联受体), GPCR(GPCR), Mini G protein(迷你G蛋白), Mini-Gs(迷你Gs)

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

  1. Expression of mini-Gs in E. coli
    Mini-Gs393 was cloned into the pET15b plasmid to allow expression in E. coli. The construct contained an N-terminal histidine tag to facilitate purification of mini-Gs and a TEV protease cleavage site to allow removal of the histidine tag (Figure 1).


    Figure 1. Mini-Gs393 E. coli expression construct. Mini-Gs was cloned into the plasmid pET15b (the sequence and map of this vector are available from the EMD Millipore website) using NcoI and XhoI restriction sites (underlined). The Mini-Gs393 construct contains an N-terminal 6x histidine tag (highlighted in red) followed by a TEV protease cleavage site (highlighted in green). Start and stop codons are shown in bold.
     
    1. Transform E. coli strain BL21-CodonPlus(DE3)-RIL with the plasmid pET15b-mini-Gs393, plate on a TYE agar plate (containing 100 μg/ml ampicillin and 34 μg/ml chloramphenicol) and incubate overnight at 37 °C. (see Note 1)
    2. Pick a single colony and inoculate 5 ml of LB media (supplemented with 0.2% glucose, 100 μg/ml ampicillin and 34 μg/ml chloramphenicol), incubate for 6-8 h at 37 °C, shaking at 220 rpm.
    3. Inoculate 150 ml of LB media (supplemented with 0.2% glucose, 100 μg/ml ampicillin and 34 μg/ml chloramphenicol) with 5 ml of the starter culture, incubate for 16-20 h at 30 °C, shaking at 220 rpm.
    4. Measure the OD600 nm of the overnight culture and inoculate 4 x 500 ml of TB media (supplemented with 0.2% glucose, 100 μg/ml ampicillin, 34 μg/ml chloramphenicol and 5 mM MgSO4) in 2 L Erlenmeyer flasks to give an OD600 nm of 0.15
    5. Incubate at 30 °C, shaking at 220 rpm, until the OD600 nm reaches 0.6-0.8 (this should take 2-3 h). Induce expression of mini-Gs by addition of IPTG to give a final concentration of 50 μM, incubate at 25 °C, shaking at 220 rpm for 16-20 h.
    6. Harvest the cells by centrifugation at 5,000 x g for 10 min at 4 °C. Flash freeze the pellet in liquid nitrogen and store at -80 °C.

  2. Purification of mini-Gs
    Mini-Gs393 is purified by Ni2+ affinity chromatography, followed by cleavage of the histidine tag using TEV protease and negative purification on Ni2+-NTA to remove the TEV and undigested mini-Gs. A final gel filtration step is performed to remove aggregated protein. This protocol yields approximately 100 mg of pure mini-Gs393 from 2 L of E. coli culture.
    1. Resuspend the pellet from 2 L of E. coli culture in buffer A to give a final volume of 200 ml.
    2. Add 4 protease inhibitor tablets, and PMSF (1 mM), Pepstatin-A (2.5 μM), Leupeptin (10 μM), DNase I (50 μg/ml), lysozyme (50 μg/ml) and DTT (100 μM) to give the final concentrations indicated.
    3. Stir the cell suspension at medium speed using a magnetic stirring bar at 4 °C for 30 min.
    4. Lyse the cells by sonication for 10 min, with pulses of 2 sec on and 4 sec off, at an amplitude of 70%, keeping the sample in an ice bath to prevent heating.
    5. Transfer the lysate to a 250 ml centrifuge bottle and centrifuge at 38,000 x g for 45 min at 4 °C.
    6. Remove the supernatant and filter through a 0.22 μm Steritop filter unit. (see Note 2)
    7. Load the supernatant onto a 10 ml HisTrap Fast Flow column (2 x 5 ml columns connected in series), pre-equilibrated with buffer A, at a flow rate of 5 ml/min at 4 °C, using a peristaltic pump.
    8. Wash the column with 100 ml of buffer B at a flow rate of 5 ml/min at 4 °C.
    9. Elute the column with 30 ml of buffer C at a flow rate of 5 ml/min at 4 °C, collecting a single 30 ml fraction.
    10. Determine the concentration of mini-Gs by measuring the A280 nm using a NanoDrop 2000 spectrophotometer. An absorbance of 1.0 unit equates to a protein concentration of 1.0 mg/ml for mini-Gs393 (extinction coefficient: 27,310 M-1 cm-1).
    11. A maximum of 200 mg of protein can be processed from this point onwards, if more mini-Gs is present it can be split into two batches and processed in parallel. (see Note 3)
    12. Add DTT to give a final concentration of 1 mM and TEV protease to give a TEV:mini-Gs ratio of 1:20 w/w. (see Note 4)
    13. Dialyse the protein against 1 L of buffer D for 2-3 h at 4 °C, discard the external buffer and dialyse against 1 L of fresh buffer D overnight at 4 °C.
    14. Transfer the protein to a 50 ml tube, add imidazole to give a final concentration of 20 mM and 4 ml of Ni2+-NTA agarose resin (pre-equilibrated with buffer D), mix on a roller mixer for 30 min at 4 °C. (see Note 5)
    15. Pour the suspension onto 1 ml of Ni2+-NTA agarose resin (pre-equilibrated with buffer D) packed into a disposable plastic column and let it run through by gravity.
    16. Collect the flow through and wash the column with 5 ml of buffer D.
    17. Pool the wash with the flow through and concentrate to 1.5 ml using an Amicon Ultra-15 concentrator (10 kDa cut-off) in a refrigerated benchtop centrifuge at 4 °C (this may take up to 2 h).
    18. Centrifuge the concentrated protein at > 15,000 x g for 10 min in a refrigerated microcentrifuge at 4 °C to remove aggregates.
    19. Load the supernatant onto a HiLoad 26/600 Superdex 200 gel filtration column (pre-equilibrated with buffer E) at a flow rate of 2.6 ml/min at 4 °C, collecting 2 ml fractions. (see Note 6)
    20. Pool peak fractions (typically 22-26 ml) based on the gel filtration chromatogram (Figure 2).
    21. Concentrate the pooled fractions to 100 mg/ml, using an Amicon Ultra-15 concentrator (10 kDa cut-off) in a refrigerated benchtop centrifuge at 4 °C (this may take up to 2 h).
    22. Determine the concentration of mini-Gs by measuring the A280 nm using a NanoDrop 2000 spectrophotometer. To ensure an accurate reading, dilute a sample of the protein 1:10 in buffer E before measuring the absorbance.
    23. Centrifuge the concentrated protein at > 15,000 x g for 10 min in a refrigerated microcentrifuge at 4 °C to remove aggregates.
    24. Transfer the supernatant to a fresh tube and discard the pellet.
    25. Aliquot 20 μl of the supernatant (2 mg of mini-Gs) into 0.5 ml tubes, flash freeze in liquid nitrogen, store at -80 °C. Mini-Gs is stable under these conditions for over 1 year.


      Figure 2. SDS-PAGE and gel filtration analysis of the mini-Gs393 purification. A. SDS-PAGE analysis of the mini-Gs393 purification. Lane: (M) molecular weight markers; (1) HisTrap column loading material (1:10 dilution); (2) HisTrap column flow through (1:10 dilution); (3) HisTrap column wash; (4) HisTrap column eluate; (5) sample after TEV digestion; (6) Ni2+-NTA negative purification flow through; (7) Ni2+-NTA negative purification eluate; (8) gel filtration pool. TEV protease is indicated by an asterisk. Note that it is not necessary to boil the samples before loading on the gel. B. Preparative gel filtration chromatogram for mini-Gs393. Pooled fractions are indicated by dashed lines, fractions at the leading edge of the peak should be omitted to ensure that aggregates are completely removed. Note that the UV detector saturates at absorbance readings above 2,700 mAu, which gives the chromatogram a truncated absorbance profile. C. Analytical gel filtration analysis of purified mini-Gs on a Superdex 200 GL 10/300 column. The chromatogram demonstrates that purified mini-Gs is monomeric (calculated molecular weight of 23 kDa compared to the theoretical value of 27 kDa) and free from aggregates.

Data analysis

Chromatograms were recorded using UNICORN software and data were plotted using GraphPad Prism 7. The analytical gel filtration column was calibrated using molecular weight standards (gel filtration marker kit), and the apparent molecular weight of mini-Gs was calculated from the calibration curve as previously published (Carpenter and Tate, 2016). No statistical analysis was performed during this work.

Notes

  1. The BL21-CodonPlus(DE3)-RIL E. coli strain contains a plasmid, carrying the chloramphenicol resistance gene, which encodes extra copies of rare tRNAs. This helps to negate the effect of codon bias when expressing eukaryotic proteins in E. coli. The standard BL21(DE3) strain can also be used for expression of mini-Gs, but in this case chloramphenicol should be omitted from all media.
  2. Filtration of the lysate significantly extends the life of the HisTrap column, which can be regenerated and reused at least 10 times.
  3. The maximum loading capacity of the HiLoad 26/600 Superdex 200 gel filtration column is approximately 150 mg of protein. TEV digestion of 200 mg of protein from the HisTrap eluate will yield approximately 150 mg of mini-Gs after the Ni2+-NTA negative purification step.
  4. TEV protease is available from commercial suppliers such as Sigma-Aldrich, but can be expensive. Alternatively, it can be easily purified in-house using any one of the protocols reported in the literature (for example, Tropea et al., 2009). The main consideration is that the purified TEV should contain a polyhistidine tag to facilitate its removal by Ni2+ negative purification after cleavage of mini-Gs.
  5. Mini-Gs has significant affinity for Ni2+ resin even after cleavage of the histidine tag. It is vital to add 20 mM imidazole at this step, otherwise mini-Gs will bind to the Ni2+ resin and will not be recovered in the flow through. This is also an important consideration for any downstream application where purified mini-Gs may contact Ni2+ resin, for example pull-down experiments.
  6. Mini-Gs tends to aggregate through nonspecific disulphide bonds, therefore TCEP is added to buffer E to maintain a reducing environment. DTT can be used instead of TCEP, but is less stable in solution.

Recipes

  1. TYE agar plates
    10 g/L tryptone
    5 g/L yeast extract
    8 g/L NaCl
    15 g/L agar
    Autoclave at 121 °C, 2.8 bar for 15 min to sterilise. Add ampicillin (100 μg/ml) and chloramphenicol (34 μg/ml) to give the final concentrations indicated
  2. Luria-Bertani (LB) media
    10 g/L tryptone
    5 g/L yeast extract
    10 g/L NaCl
    Autoclave at 121 °C, 2.8 bar for 15 min to sterilise
  3. Terrific Broth (TB) media
    24 g/L yeast extract
    12 g/L tryptone
    9.4 g/L K2HPO4
    2.2 g/L KH2PO4
    4 ml/L glycerol
    Autoclave at 121 °C, 2.8 bar for 15 min to sterilise
  4. PMSF stock solution
    200 mM in absolute ethanol, store at 4 °C
  5. Pepstatin-A stock solution
    2.5 mM in DMSO, store at -20 °C
  6. Leupeptin stock solution
    10 mM in Milli-Q water, store at -20 °C
  7. DNase I stock solution
    50 mg/ml in Milli-Q water, store at -20 °C
  8. Lysozyme Lysozyme stock solution
    50 mg/ml in Milli-Q water, store at -20 °C
  9. DTT stock solution
    1 M in Milli-Q water, make fresh
  10. GDP stock solution
    100 mM in Milli-Q water, make fresh
  11. Buffer A
    40 mM HEPES, pH 7.5
    100 mM NaCl
    10 mM imidazole
    10% v/v glycerol
    5 mM MgCl2
    50 μM GDP (add immediately before use)
    Filter using a 0.22 μm Steritop or syringe filter
  12. Buffer B
    20 mM HEPES, pH 7.5
    500 mM NaCl
    40 mM imidazole
    10% v/v glycerol
    1 mM MgCl2
    50 μM GDP (add immediately before use)
    Filter using a 0.22 μm Steritop or syringe filter
  13. Buffer C
    20 mM HEPES, pH 7.5
    100 mM NaCl
    500 mM imidazole
    10% v/v glycerol
    1 mM MgCl2
    50 μM GDP (add immediately before use)
    Filter using a 0.22 μm Steritop or syringe filter
  14. Buffer D
    20 mM HEPES, pH 7.5
    100 mM NaCl
    10% v/v glycerol
    1 mM MgCl2
    10 μM GDP (add immediately before use)
  15. Buffer E
    10 mM HEPES, pH 7.5
    100 mM NaCl
    10% v/v glycerol
    1 mM MgCl2
    1 μM GDP (add immediately before use)
    0.1 mM TCEP (add immediately before use)
    Filter using a 0.22 μm Steritop or syringe filter

Acknowledgments

This work was funded by the Wellcome Trust, Heptares Therapeutics Ltd and core funding from the Medical Research Council [MRC U105197215]. This protocol was adapted from a previously described method (Carpenter and Tate, 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, purification and crystallisation of the adenosine A2A receptor bound to an engineered mini G protein. Bio-protocol 7(8): e2234.
  4. Tropea, J.E., Cherry, S. and Waugh, D.S. (2009). Expression and purification of soluble His6-tagged TEV protease. Methods Mol Biol 498: 297-307.

简介

异源三聚体G蛋白通过将细胞表面G蛋白偶联受体(GPCR)的信息转导至细胞质效应蛋白来调节细胞内信号传导。 GPCR-G蛋白复合物的结构和功能表征对于完全破译信号转导机制是重要的。然而,当与受体偶联时,天然G蛋白质是不稳定的并具有构象的动力学。因此,我们开发了一种工程化的最小G蛋白,其与GPCR形成稳定的复合物,促进了人腺苷A 2A受体的结晶和结构测定(A 2AR)的活性构象。 Mini G蛋白是各种应用中潜在有用的工具,包括表征GPCR药理学,结合亲和力和动力学实验,激动剂药物发现和GPCR-G蛋白复合物的结构测定。在这里,我们描述了一个用于表达和纯化mini-G 的详细方案。

我们最近报告了一种工程化的最小G蛋白质(Carpenter和Tate,2016)的开发,其促进了人腺苷A 2A受体的结晶( A 2 R 2)其活性构象(Carpenter等人,2016; Carpenter和Tate,2017)。不同于需要在真核系统中表达的异源三聚体G蛋白质,在大肠杆菌(大肠杆菌)中高度表达微型G蛋白,并且可以可以容易地纯化,每升培养物的产量为50-100mg的mini-G 。在这里,我们描述了早先在Carpenter和Tate(2016)中描述的可以用于前面描述的任何一种迷你G蛋白构建体的表达和纯化的逐步方案(Carpenter等人, 2016年;卡彭特与泰特,2016年)。由于mini-G>构造393非常适合于大多数应用(参见Carpenter和Tate,2016),因此将在此使用它。

关键字:复合物, 改造的G蛋白, G蛋白偶联受体, GPCR, 迷你G蛋白, 迷你Gs

材料和试剂

  1. Steritop0.22μm过滤器(EMD Millipore,目录号:SCGPT01RE)
  2. 50ml管(SARSTEDT,目录号:62.547.254)
  3. 15 ml管(SARSTEDT,目录号:62.554.002)
  4. 2 ml管(Eppendorf,目录号:0030120094)
  5. 1.5毫升管(SARSTEDT,目录号:72.690.001)
  6. 0.5ml管(SARSTEDT,目录号:72.699)
  7. 移液器提示(STARLAB INTERNATIONAL)
  8. 塑料柱(例如,空的PD-10柱)(GE Healthcare,目录号:17043501)
  9. HisTrap Fast Flow 5 ml预填充柱(GE Healthcare,目录号:17-5255-01)
  10. Amicon Ultra-15集中器10 kDa截止(EMD Millipore,目录号:UFC901024)
  11. HiLoad 26/600 Superdex 200 PG凝胶过滤柱(GE Healthcare,目录号:28989336)
  12. SnakeSkin透析管10 kDa截止值(Thermo Fisher Scientific,Thermo Scientific TM,目录号:68100)
  13. E。大肠杆菌菌株BL21-CodonPlus(DE3)-RIL(Agilent Technologies,目录号:230245)
  14. pET15b质粒(EMD Millipore,目录号:69661)
  15. 氨苄青霉素(Melford Laboratories,目录号:A0104)
  16. 氯霉素(MP Biomedicals,目录号:0219032125)
  17. 葡萄糖(Formedium,目录号:GLU03)
  18. 七水硫酸镁(MgSO 4·7H 2 O)(VWR,目录号:25165.260)
  19. IPTG(Melford实验室,目录号:MB1008)
  20. 液氮
  21. TEV蛋白酶(内部生产)
  22. cOmplete,无EDTA的蛋白酶抑制剂片剂(Roche Diagnostics,目录号:11873580001)
  23. 溶菌酶(Sigma-Aldrich,目录号:L6876)
  24. 咪唑(Sigma-Aldrich,目录号:56748)
  25. Ni 2 + -NTA琼脂糖(QIAGEN,目录号:30210)
  26. 凝胶过滤标记试剂盒(Sigma-Aldrich,目录号:MWGF200)
  27. TCEP(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:77720)
  28. 胰蛋白胨(Melford Laboratories,目录号:GT1332)
  29. 酵母提取物(Melford Laboratories,目录号:GY1333)
  30. 氯化钠(NaCl)(Fisher Scientific,目录号:10598630)
  31. 琼脂
  32. 可怕的肉汤(TB)(Melford实验室,目录号:GT1702)
  33. 甘油(VWR,目录号:24388.320)
  34. PMSF(Sigma-Aldrich,目录号:P7626)
  35. 绝对乙醇(VWR,目录号:20821.330)
  36. Pepstatin-A(Sigma-Aldrich,目录号:P4265)
  37. DMSO(Sigma-Aldrich,目录号:D2650)
  38. 亮肽素(Sigma-Aldrich,目录号:L2884)
  39. DNase I(Sigma-Aldrich,目录号:DN25)
  40. DTT(Melford Laboratories,目录号:MB1015)
  41. GDP(Sigma-Aldrich,目录号:G7127)
  42. HEPES(Sigma-Aldrich,目录号:H3375)
  43. 氯化镁(MgCl 2)(Fisher Scientific,目录号:BP214-500)
  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. TYE琼脂平板(参见食谱)
  47. Luria Bertani(LB)媒体(见食谱)
  48. 极好的肉汤(TB)媒体(见食谱)
  49. PMSF储备溶液(见配方)
  50. 抑制素-A储备溶液(参见食谱)
  51. 亮肽酶原液(见食谱)
  52. DNase I储备溶液(参见食谱)
  53. 溶菌酶原液(见配方)
  54. DTT库存解决方案(见配方)
  55. GDP储备解决方案(见配方)
  56. 缓冲液A(参见食谱)
  57. 缓冲液B(参见食谱)
  58. 缓冲区C(见配方)
  59. 缓冲区D(见配方)
  60. 缓冲液E(参见食谱)

设备

  1. 2升锥形瓶(例如,Corning,目录号:4980-2L)
  2. 高速离心机(例如,Beckman Coulter,型号:Avanti J-26XP,目录号:393124)
  3. 磁力搅拌棒
  4. Shaker孵化器(Infors,型号:Multitron Standard)
  5. 移液器(STARLAB INTERNATIONAL)
  6. 配有13mm探针(例如Sonics Vibra-Cell)(Sonics,型号:VCX 130)的超声波仪
  7. 转杯能够旋转250毫升瓶子(例如Beckman Coulter JLA-16.250)(Beckman Coulter,目录号:363934)
  8. 蠕动泵(例如,GE Healthcare,型号:Pump P-1,目录号:18-1110-91)
  9. NanoDrop 2000分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 2000)
  10. UV/VIS分光光度计
  11. 辊式搅拌机(IKA,型号:ROLLER 6数码,目录号:0004011000)
  12. 转子能够旋转1升瓶(例如Beckman Coulter JLA-8.1000)(Beckman Coulter,目录号:363688)
  13. 冷藏式台式离心机(例如,Eppendorf,目录号:5430R)
  14. 冷冻微量离心机(例如,Eppendorf,目录号:5418R)
  15. ÄKTA净化器色谱系统(GE Healthcare,型号:ÄKTA净化器)

软件

  1. UNICORN(GE Healthcare)
  2. 图形软件(例如,Prism 7)(GraphPad)或免费替代品(例如,R生物导体封装)(生物导体)

程序

  1. 在E中表示mini-G 。大肠杆菌
    将Mini-G 393克隆到pET15b质粒中以允许在E中表达。大肠杆菌。该构建体含有N-末端组氨酸标签,以便于纯化小分子酶和TEV蛋白酶切割位点以允许去除组氨酸标签(图1)。


    图1. Mini-G 393 大肠杆菌表达构建将小型G 克隆到质粒pET15b(该载体的序列和图谱可从EMD Millipore网站获得),使用Nco I和Xho I限制性位点(下划线)。 Mini-G 393构建体含有N末端的6x组氨酸标签(以红色突出显示),随后是TEV蛋白酶切割位点(以绿色突出显示)。开始和结束密码以粗体显示。
     
    1. 变换E。大肠杆菌菌株BL21-CodonPlus(DE3)-RIL与质粒pET15b-mini-G303在TYE琼脂平板上(含有100μg/ml氨苄青霉素和34μg/ml氯霉素),并在37℃下孵育过夜。 (见注1)
    2. 挑取单个菌落并接种5ml LB培养基(补充0.2%葡萄糖,100μg/ml氨苄青霉素和34μg/ml氯霉素),37℃孵育6-8h,以220rpm摇动。 >
    3. 用5ml初始培养物接种150ml LB培养基(补充有0.2%葡萄糖,100μg/ml氨苄青霉素和34μg/ml氯霉素),在30℃下孵育16-20小时,以220rpm摇动。 br />
    4. 测量过夜培养物的OD 600nm,接种4×500ml TB培养基(补充有0.2%葡萄糖,100μg/ml氨苄青霉素,34μg/ml氯霉素和5mM MgSO4) 4μl)在2L锥形烧瓶中,得到0.15nm的OD 600nm
    5. 在30℃下孵育,以220rpm振荡,直到OD 600nm达到0.6-0.8(这应该需要2-3小时)。通过加入IPTG诱导微型G 的表达,得到最终浓度为50μM,在25℃下孵育,以220rpm摇动16-20小时。
    6. 通过在4℃下以5,000×g离心10分钟收获细胞。在液氮中将颗粒闪蒸冷冻并储存在-80°C
  2. 微型G 的净化
    通过Ni 2+亲和层析纯化Mini-G 393,然后使用TEV蛋白酶切割组氨酸标签,并在Ni 2+上纯化, sup> -NTA去除TEV和未消化的mini-G 。进行最终的凝胶过滤步骤以除去聚集的蛋白质。该方案从2L的E产生大约100毫克的纯微型Gn339。大肠杆菌文化
    1. 从2升E重悬。大肠杆菌培养在缓冲液A中,得到200ml的最终体积。
    2. 加入4个蛋白酶抑制剂片剂,PMSF(1mM),Pepstatin-A(2.5μM),亮肽素(10μM),DNase I(50μg/ml),溶菌酶(50μg/ml)和DTT(100μM)给出最终浓度。
    3. 使用磁力搅拌棒在中速下搅拌细胞悬浮液4℃30分钟
    4. 通过超声处理10分钟,脉冲为2秒,4秒,振幅为70%,将样品保持在冰浴中,以防止加热。
    5. 将裂解液转移到250ml离心瓶中,并在4℃下以38,000 x g离心45分钟。
    6. 取出上清液并通过0.22μmSteritop过滤器过滤。 (见注2)
    7. 将上清液加入到用缓冲液A预平衡的10ml HisTrap Fast Flow柱(2×5ml串联连接的柱)中,使用蠕动泵以4ml的流速以5ml/min进行。 />
    8. 用100ml缓冲液B以4ml流速5ml/min洗涤柱子。
    9. 在4℃以5ml/min的流速用30ml缓冲液C洗脱柱,收集单一的30ml级分。
    10. 通过使用NanoDrop 2000分光光度计测量A 280nm,确定mini-G 的浓度。 1.0单位的吸光度等于mini-G 393的蛋白质浓度为1.0mg/ml(消光系数:27,310M -1//sup>)。
    11. 从该点开始可以处理最多200mg的蛋白质,如果存在更多的mini-G ,则可分成两批并行处理。 (见注3)
    12. 添加DTT以得到1mM的终浓度和TEV蛋白酶,得到1:20w/w的TEV:mini-G'比值。 (见注4)
    13. 在4℃下将蛋白质与1L缓冲液D进行透析2-3小时,弃去外部缓冲液,并在4℃下将1L新鲜缓冲液D透析过夜。
    14. 将蛋白质转移到50ml管中,加入咪唑至最终浓度为20mM,加入4ml Ni 2 O 3 -NTA琼脂糖树脂(用缓冲液D预平衡),在辊上混合在4℃下搅拌30分钟。 (见注5)
    15. 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D预平衡),并使其通过重力流过。
    16. 收集流量并用5ml缓冲液D洗涤柱
    17. 在4℃的冷藏台式离心机中使用Amicon Ultra-15浓缩器(10kDa截留)浓缩至1.5ml浓缩液(这可能需要2小时)。
    18. 将浓缩的蛋白质离心>在4℃的冷冻微量离心机中将15,000×g 10分钟,以除去聚集体。
    19. 将上清液加载到HiLoad 26/600 Superdex 200凝胶过滤柱(用缓冲液E预平衡)中,流速为2.6ml/min,4℃,收集2ml级分。 (见注6)
    20. 基于凝胶过滤色谱的池峰分数(通常为22-26ml)(图2)
    21. 使用Amicon Ultra-15浓缩器(10 kDa截止值)在4°C的冷冻台式离心机中将合并的馏分浓缩至100mg/ml(这可能需要2 h)。
    22. 通过使用NanoDrop 2000分光光度计测量A 280nm,确定mini-G 的浓度。为了确保读数的准确性,在测量吸光度之前,稀释缓冲液E中蛋白质1:10的样品
    23. 将浓缩的蛋白质离心>在4℃的冷冻微量离心机中将15,000×g 10分钟,以除去聚集体。
    24. 将上清液转移到新鲜管中,弃去沉淀。
    25. 将等分试样将20μl上清液(2mg mini-G ss)分装入0.5ml管中,在液氮中闪蒸冷冻,储存在-80℃。在这些条件下,Mini-G 在1年以上是稳定的。


      图2.微型Gs393纯化的SDS-PAGE和凝胶过滤分析。A.迷你G 393纯化的SDS-PAGE分析。泳道:(M)分子量标记; (1)HisTrap柱装载材料(1:10稀释); (2)HisTrap柱流过(1:10稀释); (3)HisTrap柱洗; (4)HisTrap柱洗脱液; (5)TEV消化后样品; (6)Ni 2 + -NTA负净化流通; (7)Ni 2 + -NTA阴性纯化洗脱液; (8)凝胶过滤池。 TEV蛋白酶由星号表示。注意,在将样品加载到凝胶之前不需要煮沸。 B.用于mini-G 393的制备性凝胶过滤色谱图。合并的馏分用虚线表示,峰值前缘的馏分应被省略,以确保聚集体被完全去除。请注意,紫外检测器在吸光度读数高于2,700 mAu时饱和,这使色谱图具有截断的吸光度分布。 C.在Superdex 200GG 10/300柱上的纯化的mini-G 的分析凝胶过滤分析。色谱图表明,纯化的小分子是单体的(计算分子量为23kDa,理论值为27kDa),不含聚集体。

数据分析

使用UNICORN软件记录色谱图,并使用GraphPad Prism 7绘制数据。使用分子量标准品(凝胶过滤标记试剂盒)校准分析凝胶过滤柱,并且从如前所述的校准曲线计算mini-Gs的表观分子量出版(Carpenter和Tate,2016)。在这项工作中没有进行统计分析。

笔记

  1. BL21-CodonPlus(DE3)-RIL E。大肠杆菌菌株含有携带氯霉素抗性基因的质粒,其编码稀有tRNA的额外拷贝。这有助于否定在E中表达真核蛋白时密码子偏倚的影响。大肠杆菌。标准BL21(DE3)菌株也可用于表达mini-G ,但在这种情况下,所有培养基中都应省略氯霉素。
  2. 裂解物的过滤显着延长了HisTrap柱的寿命,可以再生和重复使用至少10次。
  3. HiLoad 26/600 Superdex 200凝胶过滤柱的最大负载量约为150 mg蛋白质。来自HisTrap洗脱液的200mg蛋白质的TEV消化将在Ni 2 + -NTA负净化步骤之后产生大约150mg的迷你-G 。
  4. TEV蛋白酶可从商业供应商如Sigma-Aldrich获得,但可能是昂贵的。或者,可以使用文献中报道的任何一种方案(例如,Tropea等人,2009),在内部容易地纯化。主要考虑的是,纯化的TEV应包含多组氨酸标签,以便在切割迷你G 后通过Ni 2 + 阴性纯化除去。
  5. 即使在组氨酸标签切割后,Mini-G 对Ni 2 + 树脂也具有显着的亲合力。在该步骤中加入20mM咪唑是至关重要的,否则mini-G 将结合到Ni 2 +树脂上,并且不会在流过中回收。这对于其中纯化的微型G可以接触Ni 2+树脂的任何下游应用也是重要的考虑因素,例如下拉实验。
  6. Mini-G 倾向于通过非特异性二硫键进行聚合,因此将TCEP加入到缓冲液E中以维持还原环境。可以使用DTT代替TCEP,但是在解决方案中不太稳定

食谱

  1. TYE琼脂板
    10g/L胰蛋白胨
    5克/升酵母提取物
    8克/升NaCl
    15 g/L琼脂
    高压灭菌121°C,2.8 bar 15分钟灭菌。加入氨苄青霉素(100μg/ml)和氯霉素(34μg/ml),得到最终浓度指示
  2. Luria-Bertani(LB)媒体
    10g/L胰蛋白胨
    5克/升酵母提取物
    10g/L NaCl
    高压灭菌121°C,2.8 bar 15分钟消毒
  3. 极好的肉汤(TB)媒体
    24 g/L酵母提取物
    12克/升胰蛋白胨
    9.4g/L K 2 HPO 4
    2.2g/L KH PO 4
    4 ml/L甘油
    高压灭菌121°C,2.8 bar 15分钟消毒
  4. PMSF库存解决方案
    无水乙醇200mM,储存于4℃
  5. 消化抑素-A储备溶液
    在DMSO中2.5mM,储存于-20℃
  6. 亮色抑素储备液
    在Milli-Q水中10 mM,储存于-20°C
  7. DNase I库存解决方案
    在Milli-Q水中50 mg/ml,储存于-20°C
  8. 溶菌酶溶菌酶原液
    在Milli-Q水中50 mg/ml,储存于-20°C
  9. DTT库存解决方案
    1米在Milli-Q水中,使新鲜的
  10. GDP库存解决方案
    100毫升在Milli-Q水中,使新鲜的
  11. 缓冲区A
    40 mM HEPES,pH 7.5
    100 mM NaCl
    10 mM咪唑
    10%v/v甘油
    5mM MgCl 2
    GDP(在使用前立即添加)
    使用0.22微米的Steritop或注射器过滤器过滤
  12. 缓冲区B
    20 mM HEPES,pH 7.5
    500 mM NaCl
    40 mM咪唑
    10%v/v甘油
    1mM MgCl 2
    GDP(在使用前立即添加)
    使用0.22微米的Steritop或注射器过滤器过滤
  13. 缓冲区C
    20 mM HEPES,pH 7.5
    100 mM NaCl
    500毫克咪唑
    10%v/v甘油
    1mM MgCl 2
    GDP(在使用前立即添加)
    使用0.22微米的Steritop或注射器过滤器过滤
  14. 缓冲区D
    20 mM HEPES,pH 7.5
    100 mM NaCl
    10%v/v甘油
    1mM MgCl 2
    GDP(增加使用前立即添加)
  15. 缓冲区E
    10mM HEPES,pH7.5
    100 mM NaCl
    10%v/v甘油
    1mM MgCl 2
    GDP 1%(使用前立即添加)
    0.1 mM TCEP(使用前立即添加)
    使用0.22微米的Steritop或注射器过滤器过滤

致谢

这项工作由Wellcome Trust,Heptares Therapeutics Ltd和医学研究委员会(MRC U105197215)的核心资金资助。该方案从前面描述的方法(Carpenter和Tate,2016)改编。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. 木匠,B.和泰特,C.G. (2017)。腺苷的表达,纯化和结晶与工程化的迷你G蛋白结合的2A 2A受体。生物协议7(8):e2234。
  4. Tropea,JE,Cherry,S.and Waugh,DS(2009)。  可溶性His6标记的TEV蛋白酶的表达和纯化。方法Mol Biol 498:297-307。
  • English
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
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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