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May 2016

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Expression and Purification of Yeast-derived GPCR, Gα and Gβγ Subunits for Structural and Dynamic Studies
重组酵母的GPCR、Gα和Gβγ亚基的表达和纯化,用于结构和动力学研究   

Wenjie  ZhaoWenjie Zhao*Xudong  WangXudong Wang*Libin YeLibin Ye  (*共同第一作者)
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Abstract

In the last several years, as evidence of a surged number of GPCR-G complex structures, the expressions of GPCRs and G proteins for structural biology have achieved tremendous successes, mostly in insect and mammalian cell systems, resulting in more than 370 structures of over 70 GPCRs have been resolved. However, the challenge remains, particularly in the conformational transition and dynamics study area where a much higher quantity of the receptors and G proteins is required even in comparison to X-ray and cryo-EM (5 mg/ml, 3 μl/sample) when NMR spectroscopy (5 mg/ml, 250 μl /sample) is applied. As a result, the expression levels of the insect and mammalian systems are also difficult to meet this demand, not to mention the prohibitive cost of producing GPCRs and G proteins using these systems for a vast majority of laboratories. Therefore, exploration of an effective, affordable, and practical approach with broad applicability is demanded. Pichia pastoris expression system has shown its promise in the GPCR preparation with many merits that other eukaryotic expression systems can’t compete with. GPCRs expressed in this system are inexpensive, easy-to-manipulate, and capable of isotopically labeling. Herein, we present related protocols recently developed and upgraded in our lab, including expressions and purifications of P. pastoris derived GPCR along with Gα and Gβγ proteins. We anticipate that these protocols will advance the conformational transition and dynamics studies of the GPCR and its complexes.

Keywords: GPCR (G蛋白偶联受体), G proteins (G蛋白), Yeast (酵母), Conformation (构造), Dynamics (动力学), FPLC (快速蛋白液相色谱), High-yield Productivity (高收益的生产力)

Background

G protein-coupled receptor (GPCR), the biggest membrane protein family, plays critical roles in many (patho) physiological activities. Dysfunctions of GPCRs or their effectors will result in various pathologies, including neuro-degenerative diseases, cancers, and chronic inflammations (Overington et al., 2006). Although more than 350 structures (Congreve et al., 2020) of over 70 GPCRs have been resolved in complex with ligands and/or transducins, our understanding of how the signaling is propagated through the receptor upon ligand binding to downstream signaling partners is still elusive, in part due to the resolved structures merely reflect static snapshots of the dynamics signaling processes for these complexes. In addition, limitations of the structures themselves include missing parts of flexible domains and side-chain information of many residues that were not able to be resolved in both X-ray and cryo-EM spectroscopies. The development of complementary approaches to acquire the missing information would provide invaluable insights that broaden our understanding of the sequential signaling process of the GPCRs and guide us to the discovery of drugs that target specific malfunctional signaling.


Obviously, NMR is a superb tool to interrogate the conformational transition and dynamics of the GPCRs and their signaling partners with minimal structure-function perturbation beyond static structures. In the NMR study, a wild-type or minimally modified construct is often used, which can maximally reflect the genuine behaviors of the receptor when conducted in a mimic physiological environment such as detergent MNG-3, micelle, or high-density lipoprotein particle (HDL) systems after reconstitution. The specific requirements for the NMR study also propose the challenges for a regular expression system such as E. coli heterogenous expression system. Although it is the simplest and most efficient system, the expressed proteins (especially human-derived ones) lack post-translational modifications, which often results in the loss of functionality. Insect cells such as sf9, sf21, or Hi5, and mammalian expression systems such as HEK293 and CHO cells are increasingly used for structural biology especially for X-ray and cryo-EM spectroscopic studies where a relatively small quantity of the receptors is needed. However, for NMR study, the need for a large protein quantity along with isotopic labeling makes insect and mammalian systems challenging to be employed in this type of study. Therefore, we developed a yeast-based expression system to produce receptors and their transducins meet this requirement. In this protocol, we are particularly focused on a broadly applicable strategy of preparing GPCRs, Gα and Gβγ, in which the protocol for GPCR preparation is further upgraded based on our previous publications (Ye et al., 2016, 2018a and 2018b) with a significantly improved productivity. The Gα and Gβγ preparations have been incorporated into this set of protocols as a complete system for GPCR-G protein complex study. The strategy described in the manuscript can also be applied to other signaling partners such as GRKs and β-arrestins. It is also easy to switch to isotopically labeling if needed. The advantages of expressing these receptors and effectors in the P. pastoris system are obvious in comparison with other systems aforementioned.


Materials and Reagents

  1. 1.5 ml microcentrifuge tubes (Fisher Scientific, catalog number: 05-408-130)

  2. 2 mm electroporation cuvette (Fisher Scientific, catalog number: FB102)

  3. Nitrocellulose membrane (Bio-Rad, catalog number: 1620112)

  4. NalgeneTM Rapid-FlowTM Sterilize Disposable Filter Units with Nylon Membrane (Thermo Scientific, catalog number: 09-740-24A)

  5. FisherbrandTM bulk-packed HDPE 7 ml scintillation vial (Fisher Scientific, catalog number: 03-337-20)

  6. WhatmanTM binder-free glass microfiber filters, grade GF/C (Cytiva, catalog number: 09-874-32A)

  7. DWK Life Sciences KimbleTM solid borosilicate glass beads (Fisher Scientific, catalog number: 10-310-3)

  8. PuritanTM hospital standard cotton swab (Fisher Scientific, catalog number: 22-029-684)

  9. SartorisTM VivaspinTM 20 centrifugal concentrator (Sartorius, catalog number: 14-558-501)

  10. Thermo ScientificTM NalgeneTM Rapid-FlowTM sterilize disposable filter units with nylon membrane (Fisher Scientific, catalog number: 0974024A)

  11. Slide-A-LyzerTM MINI Dialysis Devices, 3.5K MWCO, 0.5 ml (Fisher Scientific, catalog number: 88400)

  12. 14 ml round-bottom tubes (Fisher Scientific, catalog number: 12-565-971)

  13. 250 ml Nalgene® centrifuge bottles (Sigma-Aldrich, catalog number: Z353736)

  14. Pichia pastoris SMD 1163 strain (Invitrogen)

  15. pPIC9K vector (Invitrogen)

  16. pPIC9K_ADORA2A (Engineered from pPIC9K)

  17. XL-10-GoldTM ultracompetent E. coli cells (Agilent, catalog number: 200314)

  18. BCA Protein Assay Kit (Pierce, catalog number: PI23227)

  19. Imidazole (Fisher Chemical edge, catalog number: 03196-500)

  20. EDTA (Sigma-Aldrich, catalog number: E5134)

  21. Glacial acetic acid (Fisher Scientific, catalog number: A385-500)

  22. Methanol (Fisher Scientific, catalog number: 412-500 or A412P4)

  23. Glycerol (Fisher Scientific, catalog number: G33-500)

  24. D-sorbitol (Fisher Scientific, catalog number: S459)

  25. G418 sulfate (Fisher Scientific, catalog number: BP6735 or Wisent Inc., catalog number: 400-130-XG)

  26. Ampicillin sodium salt (Wisent Inc., catalog number: 400-110-EG or Fisher Scientific, catalog number: BP176025)

  27. LB miller broth (Wisent Inc, catalog number: 800-061-1K)

  28. LB agar miller powder (Fisher Scientific, catalog number: BP14252)

  29. Yeast extract (Wisent Inc., catalog number: 800-150-IK)

  30. Bacteriological peptone (Wisent Inc., catalog number: 800-157-IK)

  31. Yeast nitrogen base w/o amino acid (Wisent Inc., catalog number: 800152018WG)

  32. Dextrose (D-Glucose) (Fisher Scientific, catalog number: D16-500)

  33. Agar (Fisher Scientific, catalog number: BP1423-500)

  34. D-Biotin (Sigma-Aldrich, catalog number: 2031)

  35. L-histidine (Sigma-Aldrich, catalog number: H8000-5G or Fisher Scientific, catalog number: BP382100)

  36. DMSO (Fisher Scientific, catalog number: D1391)

  37. Theophylline (Sigma-Aldrich, catalog number: T1633)

  38. NaCl (Fisher Scientific, catalog number: S271-500)

  39. Bis-Tris (Fisher Scientific, catalog number: BP301-500)

  40. Lauryl maltose neopentyl glycol (MNG-3) (Anatrace, catalog number: MG310)

  41. Cholesterol hemisuccinate (CHS) (Sigma-Aldrich, catalog number: C6512)

  42. Talon Metal Affinity Resin (TaKaRa, catalog number: 635503)

  43. (Optional) Liquid Nitrogen

  44. BamHI-HF (New England BioLabs, catalog number: R3136S)

  45. NotI-HF (New England BioLabs, catalog number: R3189S)

  46. PmeI (New England BioLabs, catalog number: R0560S)

  47. T4 DNA ligase (New England BioLabs, catalog number: M0202S)

  48. Quick CIP (New England BioLabs, catalog number: M0525S)

  49. GenEluteTM Plasmid Miniprep Kit (Sigma-Aldrich, catalog number: PLD35-1KT)

  50. 100% ethanol (Fisher Scientific, catalog number: A4094)

  51. HRP-conjugated 6*His, His-Tag Monoclonal Antibody (Proteintech, catalog number: HRP-66005)

  52. DDDDK TAG Polyclonal Antibody (Equivalent to FLAG® Antibodies) (Proteintech, catalog number: HRP-20543-1-AP)

  53. Zymolyase-20T (Amsbio, catalog number: 120493-1)

  54. Xanthine amine congener (Sigma-Aldrich, catalog number: X103)

  55. Affi-GelTM 10 (Bio-Rad, catalog number: 1536099)

  56. Isopropanol (Fisher Scientific, catalog number: A426P-4)

  57. Antifoam A Concentrate (Sigma-Aldrich, catalog number: A5633)

  58. Thermo ScientificTM Yeast DNA Extraction Kit (Fisher Scientific, catalog number: PI78870)

  59. Thermo ScientificTM Y-PERTM Yeast Protein Extraction Reagent (Fisher Scientific, catalog number: PI78991)

  60. Universal-ES liquid scintillation cocktail (MP BiomedicalsTM, catalog number: 88248001)

  61. 3,3’,5,5’-tetramethylbenzidine solution (Alfa Aesar, catalog number: J61325)

  62. PageRuler Plus Prestained Protein Ladder (Fisher Scientific, catalog number: 26619)

  63. BioAcryl-P (30%, 29:1) liquid (Alfa Aesar, catalog number: J63279)

  64. QIAquick Gel Extraction Kit (Qiagen, catalog number: 28704)

  65. 10× Phosphate buffer (see Recipes)

  66. Immunoblotting Blocking buffer (see Recipes)

  67. Immunoblotting Incubation buffer (see Recipes)

  68. Immunoblotting Washing buffer (see Recipes)

  69. LB plates (see Recipes)

  70. YNBD plates (see Recipes)

  71. YPD medium (see Recipes)

  72. YPD agar plates (see Recipes)

  73. BMGY medium (see Recipes)

  74. BMMY medium (see Recipes)

  75. Common Washing Buffer P1 (see Recipes)

  76. Receptor Lysis Buffer P2 (see Recipes)

  77. Receptor Preparation Buffer P3 (see Recipes)

  78. Receptor Preparation Column Washing Buffer P4 (see Recipes)

  79. Column Elution Buffer P5 (see Recipes)

  80. XAC Column Elution Buffer P6 (see Recipes)

  81. G Protein Washing Buffer P1 (see Recipes)

  82. G Protein Lysis buffer P2 (see Recipes)

  83. G Protein Elution Buffer P3 (see Recipes)

  84. Q Buffer A-Gα (see Recipes)

  85. Q Buffer B-Gα (see Recipes)

  86. Q Buffer A-Gβγ (see Recipes)

  87. Q Buffer B-Gβγ (see Recipes)

  88. Immunoblotting Blocking Buffer (see Recipes)

  89. Immunoblotting Washing Buffer (see Recipes)

  90. Radioligand Binding/Washing Buffers (see Recipes)

Equipment

  1. (Optional) -80 °C freezer

  2. Thermo ScientificTM SorvallTM Centrifuge (Thermo Scientific, model: ST 40R)

  3. EppendorfTM Microcentrifuge (Eppendorf, model: Legend Micro 21R)

  4. Applied BiosystemsTM SimpliAmpTM Thermal Cycler (Applied Biosystems, model: A24812)

  5. GenesysTM UV-vis spectrometer (Genesys, model: 10S)

  6. Thermo ScientificTM Heratherm Microbiological Incubator 117L (Thermo Scientific, model: IGS100)

  7. NanoDropTM spectrophotometer (NanoDrop, model: Lite)

  8. Beckman L8-70M ultracentrifuge (Beckman, model: L8-70M)

  9. ÄKTApurifier FPLC (GE, model: Purifier 100)

  10. Cell electroporation system electroporator (Bio-Rad, model: Gene Pulser X)

  11. FisherbrandTM Heavy-Duty Vortex Mixer (Fisherbrand, model: STD)

  12. 5 L New Brunswick bioreactor (New Brunswick, model: BIOFLO III)

  13. MicrofluidizerTM Processor (IDEX Material Processing, model: LM20)

Procedure

This protocol was modified and finalized based on our prior (Ye et al., 2016 and 2018b) as well as current work. Different from our previous work, all target genes presented in this protocol were codon optimized using online codon optimization software from the IDT website and respective genes were synthesized prior to integration into the genome of P. pastoris via BamHI and NotHI restriction enzyme. In particular, both human derived adenosine A2AR (35.1KDa) and A1 receptor (36.5KDa) genes had FLAG and His10 tags in the N- and C-terminal ends, respectively. In addition, following the FLAG tag, a TEV protease cleavage site as well as α-Factor peptide were added in the front of genes (Figure 1). In contrast, there was no FLAG tag and α-Factor sequences in Gα (45.7KDa) as well as Gβ (38.7KDa) and Gγ (7.6KDa) constructs; in particular, there was no His-tag sequence in Gγ considering Gβ and Gγ were co-expressed together.



Figure 1. Constructs for GPCRs and G proteins described in this protocol. The pPIC9K-A1R/pPIC9K-A2AR had α-Factor peptide, the FLAG tag, a TEV protease cleavage. In contrast, there was no FLAG tag and α-Factor sequences in the G protein constructs; in particular, there was no His-tag motif in Gγ considering Gβ and Gγ were co-expressed together.


  1. Plasmid Preparation for GPCRs and G proteins

    1 μl of the construct pPIC9K_ADORA2A which was a recombinant vector of pPIC9K containing the ADORA2A receptor sequence, in a concentration of ~100 ng/μl, generously provided by T. Kobayashi (Japan) (Andre et al., 2006), was chemically transformed into 2 μl XL-10 Gold competent cells by heat shock for 45 s at 42 °C.

    1. 200 μl of LB medium was immediately added into the mixture and the transformed cells were spread on the 25 ml LB plates containing 50 μg/ml ampicillin. Incubated at 37 °C overnight.

    2. One colony was picked from the plate and inoculated into a 4 ml LB medium containing 50 μg/ml ampicillin and cultured overnight at 37 °C.

    3. The plasmid was extracted using GenEluteTM Plasmid Miniprep Kit following the instruction.

    4. The extracted plasmids were digested with 1 μl BamHI-HF and1 μl NotI-HF with 1× NEB CutSmart® Buffer for 1.5 h in a 1.5 ml Eppendorf tube. 1 μl Quick CIP was added for additional 30 min digestion and phosphorylation to create pPIC9K backbone bearing BamHI and NotI restriction sites on each side. The total volumes were varied on the amounts of plasmids used. Usually, 1 μl of each restriction enzyme was used for the digestion of 20 μg plasmids.

    5. All target gene fragments including A2AR, A1R, Gαs, Gβ, and Gγ (~200 ng/μl) were synthesized in accordance with P. pastoris codon optimized sequences. Of note, all gene fragments beared BamHI and NotI restriction enzyme sites at N- and C-termini.

    6. The gene fragments were also digested with 1 μl of each BamHI-HF and NotI-HF restriction enzymes with 1× NEB CutSmart® Buffer for 2 h without Quick CIP treatment.

    7. Both digested pPIC9K plasmid and gene fragments were run DNA electrophoresis (Krettler et al., 2013) and purified using QIAquick Gel Extraction Kit and the concentrations were measured using NanoDropTM Lite spectrophotometer.

    8. The ligation process was processed with various ratios between backbone plasmid and gene fragments using 1 μl T4 DNA Ligase with 1× NEB T4 DNA Ligase Buffer in a 0.2 ml PCR tube. The molar ratios between gene fragments and empty plasmids were set to 1:3, 1:1, and 3:1 with concentrations of 10-100 ng/μl.

    9. The ligated plasmids containing different gene fragments were transformed into XL-10 Gold competent cell (Smbrook et al., 1989; Dubnau, 1999) and spread on the LB plates containing 50 μg/ml ampicillin. Incubated overnight at 37 °C.

    10. The plasmids for each constructs were extracted using GenEluteTM Plasmid Miniprep Kit. 20 μg plasmid in 100 μl for each construct was linearized with 1 μl PmeI restrict enzymes with 1× NEB CutSmart® Buffer for 2 h in a 0.2 ml PCR tube.

    11. 200 μl of 100% ethanol was added into the 100 μl linearized plasmids and incubated on the ice for 5 min.

    12. The linearized plasmid was then centrifuged for 5 min at a speed of 16,200 × g at 4 °C.

    13. The supernatant was discarded, and the linearized DNA pellet was dried under a Fume hood for 20 min before re-suspended in 20 μl of distilled water with DNA concentration around 1 μg/μl.

    14. 10 μl of linearized plasmids were mixed gently with 80 μl P. pastoris competent cells in 1.5 ml Eppendorf tube and kept on ice for 5 min prior to transfer into a 2 mm electroporation cuvette.

    15. The transformations were performed using a Gene Pulser II electroporation with the condition of 1,500 V charging voltage, 25 μF capacitance, and 400 Ω resistance.

    16. 1 ml of ice-cold 1 M sorbitol was immediately added into the electroporation cuvette and transferred into 14 ml round-bottom tubes.

    17. The samples were then incubated for 3 h at 30 °C without shaking prior to spreading them onto YNBD plates.

    18. Of note, the linearized Gβ and Gγ plasmids (10 μl each) were transformed into the P. pastoris together while all other linearized plasmids were transformed separately.


  2. Preparation of High-yield Constructs for GPCRs and G Proteins

    As shown in Figure 2A, the colonies grown on the YNBD plates after 3-5 days incubation were transferred onto YPD plates containing 1 mg/ml G418 and incubated 3-5 days at 30 °C. YPD plates with gradient G418 concentration were prepared and stored at 4 °C for subsequent screening.



    Figure 2. Schematic procedure of electroporation and high-yield construct screening for adenosine A2AR and A1R receptors (A) and their identifications using SDS-PAGE and western blotting assays (B)


    1. The colonies grown on YPD plates containing 1 mg/ml G418 were further transferred onto second YPD plates containing 2 mg/ml G418. Incubated 3-5 days at 30 °C.

    2. Consecutively, the colonies grown on YPD plates containing 2 mg/ml G418 were finally transferred onto YPD plates containing 4 mg/ml G418. Incubated additional 3-5 days at 30 °C.

    3. 5-10 colonies on YPD plates containing 4 mg/ml G418 were picked for expression level evaluation using the immunoblotting assays against His10 and FLAG tags. Multiple integrated copies of pPIC9K can increase the Geneticin® resistance level from 0.5 mg/ml (1-2 copies) up to 4 mg/ml (7-12 copies).

    4. The single colonies were inoculated into 4 ml BMGY medium in 14-ml Falcon tubes at 30 °C for at least 24 h with shaking (275 rpm) until OD595 = 2.0-6.0.

    5. The medium was then transferred into 200 ml BMMY medium in 500 ml shake flasks covered by a cotton plug.

    6. The cells were continued culturing 60 h at 22 °C with shaking at 275 rpm.

    7. 0.5% methanol/12 h was added into the medium in order to induce and maintain the receptor or G protein expression.

    8. At the end of induction, the cell pellets were collected after centrifugation at 3,800 × g for 10 min at 4 °C in 250 ml centrifuge bottles.

    9. The cell pellets were washed once with Common Washing Buffer P1 in a ratio of 1:2.

    10. The cell pellets were then resuspended into Receptor Lysis Buffer P2 using the ratio of cell pellets to buffer equal to 1:4.

    11. The cell pellets suspensions were then vortexed at 2,000 rpm for 2 h at 4 °C in the presence of a slurry of 5 mm glass beads.

    12. The disrupted cell pellets were centrifuged at 9,720 × g for 30 min at 4 °C and the unbroken cells and cellular debris were discarded.

    13. The supernatant containing cell membrane was collected and applied to immunoblotting assays (Gallagher and Chakavarti, 2008).

    14. For accuracy, immunoblotting was performed for both anti-His and anti-FLAG in response to the FLAG-tag and His-tags, respectively.

    15. 1 μl of supernatant was blotted on nitrocellulose membrane and allowed to dry.

    16. A second 1 μl of supernatant was applied onto the previous position and let it dry.

    17. The membrane was placed in 20 ml Immunoblotting Blocking Buffer for 1 h at room temperature.

    18. The membrane was then transferred to 20 ml Immunoblotting Incubation Buffer containing either His-tag antibody (1:2,000) or FLAG-tag antibody (1:2,000) for 2 h.

    19. The membrane was then washed three times with Immunoblotting Washing Buffer, followed by distilled water.

    20. The membrane was finally visualized by BM Blue POD substrate (3,3’,5,5’-tetramethylbenzidine solution).

    21. The strongest intensity colonies were selected for further expressions. If the expression level was less than 0.5 mg/L cell culture, a second-cycle plasmid transformation was conducted following the instruction described in Procedure A and B. The colonies were directly screened on YPD plates containing 6 mg/ml G418 (Figure 2A).

    22. The stocks of screened high-yield expression constructs were made with 20% autoclaved glycerol and frozen in the -80 °C freezer or Liquid Nitrogen.


  3. Expression of GPCRs and G proteins

    The expressions of GPCRs and G proteins were using the similar procedure with slight variances described in the following section.

    1. Glycerol stocks of the transformants were inoculated onto YPD agar plates containing 0.1 mg/ml G418.

    2. 3-5 days later, a single colony was inoculated into 4 ml autoclaved YPD at 30 °C in 14 ml round bottom tubes with shaking at 275 rpm for 24 h.

    3. 4 ml medium was subsequently transferred into 200 ml autoclaved BMGY medium and cultured at 30 °C in 500 ml shake flask covered by aluminum foil with a shaking speed of 275 rpm till OD595 reached 2-6, which would take about 24 h.

    4. To induce expression, 200 ml cell pellets were spun down and transferred into 1 liter of autoclaved BMMY medium in 2.8 L flasks and cultured at 20 °C with a shaking speed of 275 rpm. Filtered methanol was added every 12 h with 0.5% (5 ml/L medium) if the baffled flasks were used. If the fermenter was used, the rate of methanol addition was controlled at maximal 1 ml/h for each liter, which was dependent on the culture volume

    5. The cells for receptors expression were collected after 80 h fermentation while the cells for G proteins expression were collected after 60 h.


  4. Purifications of GPCRs and G proteins

    Purifications of GPCRs, Gα, and Gβγ proteins were different in this protocol. Therefore, we described them separately in the following sections.


    Purification of GPCRs

    1. After 80 h expression, the cell pellets were collected at 3,800 × g for 10 min at 4 °C in 250 ml centrifuge bottles.

    2. Cell pellets were washed once with Common Washing Buffer P1 in the ratio of 1 g cell pellets to 2 ml P1 and centrifuged at 3,800 × g for 10 min at 4 °C in 250 ml centrifuge bottles.

    3. The washed cell pellets were resuspended in Receptor Lysis buffer P2 in a ratio of 4:1 to ensure the suspension was sufficient in 250 ml centrifuge bottles.

    4. The cells were then disrupted using the Microfluidizer for four cycles on the ice at the working pressure of 15,000 psi. The Microfluidizer was balanced with buffer P2 prior to the lysis.

    5. Intact cells and cell debris were separated from the membrane suspension at 9,720 × g for 30 min at 4 °C in 250 ml centrifuge bottles.

    6. The supernatant was then collected and centrifuged at 100,000 × g for 75 min using Type 45 Ti rotor for the Beckman Ultracentrifuge with corresponding tubes.

    7. The supernatant from ultracentrifugation was discarded and the membranes from different runs were collected together and suspended in Common Washing Buffer P1 to remove the EDTA.

    8. The supernatant from ultracentrifugation was discarded and the membrane pellets were dissolved in a 50 ml conical centrifuge tube containing Receptor Preparation Buffer P3 and shaking at 4 °C until all membranes were dissolved sufficiently.

    9. The solution was centrifuged at 1,980 × g for 5 min at 4 °C to remove the undissolved membrane.

    10. The dissolved membranes were mixed with pre-balanced Talon Resin using Receptor Preparation Column Washing Buffer P4. Usually, 2 ml Talon Resin was used for 6 g membrane. Incubated for 2 h at 4 °C. During the incubation, the imidazole was added to the final concentration of 100 μM.

    11. Two hours later, the Talon resin was packed onto a disposal column and washed with 5 column volumes of Receptor Preparation Column Washing Buffer P4.

    12. Subsequently, the receptors were eluted from the column using the Column Elution Buffer P5 at a gravity rate.

    13. The eluted receptors were concentrated to 1 ml by Ultra-15 Centrifugal filters 3K and buffer change with 10 ml Receptor Preparation Column Washing Buffer P4 one time with a dilution factor of 10 at 4 °C with the speed of 3,846 × g.

    14. The receptor then went through the XAC ligand column, which was pre-balanced using Receptor Preparation Column Washing Buffer P4. Repeated three times.

    15. Wash the receptor bound XAC column for 2 column volumes using Receptor Preparation Column Washing Buffer P4 to remove the non-functional receptors.

    16. The receptors were then eluted out using XAC Column Elution Buffer P6 consisting of Receptor Preparation Column Washing Buffer P4 with 25 mM theophylline.

    17. The eluted receptors were concentrated into 1 ml by Ultra-15 Centrifugal filters 3K and dialyzed against Receptor Preparation Column Washing Buffer P4 by Slide-A-LyzerTM MINI Dialysis Devices 3.5K with a dilution factor of 106 to remove all ligands to bring the receptor into the apo state.

    18. The purified receptors usually with 0.5-2 mg/L productivity were validated by SDS-PAGE (Laemmli, 1970) as well as immunoblotting, as shown in Figure 2B.


    Purification of Gα
    1. Cell pellets were harvested by centrifugation at 3,800 × g for 10 min using the centrifuge at 4 °C in 250 ml centrifuge bottles.

    2. Cell pellets were washed once with Common Washing Buffer P1 in the ratio of 1 g cell pellets to 2 ml P1 and centrifuged with the speed of 3,800 × g for 10 min at 4 °C in 250 ml centrifuge bottles.

    3. Cell pellets were resuspended in G Protein Lysis buffer P2 in a ratio of 1:4.

    4. Cells were broken by Microfluidizer using 4 cycles at 15,000 psi for completely disrupting the yeast cell wall. The Microfluidizer was balanced with buffer P2 prior to the lysis.

    5. The lysate was centrifuged at 4 °C for 30 min for 9,720 × g in 250 ml centrifuge bottles.

    6. The supernatant was applied to Talon resins for 2 h, in which imidazole was added with a final concentration of 100 μM in order to decrease non-specific binding.

    7. The G protein bound Talon resins were applied to a disposal column.

    8. The packed column was then washed with 5 column volumes of G Protein Washing buffer P1.

    9. The target Gα was eluted with 10 ml G Protein Elution Buffer P3 at a gravity rate. Strategically recycling the elution buffer to reduce the elution volume was optional.

    10. MgCl2 was added to the final concentration of 1 mM as well as GDP of 50 μM.

    11. The Gα was concentrated to 2 ml by Ultra-15 Centrifugal filters, 3K at 4 °C with the speed of 3,846 × g. The FPLC system was balanced with Buffer A- Gα for Q Sepharose Column prior to applying the eluted Gα protein into the system.

      1. Q Buffer A- Gα: 50 mM HEPES, pH 8.0, 50 μM GDP, 1 mM MgCl2.

      2. Q Buffer B- Gα: 50 mM HEPES, pH 8.0, 50 μM GDP, 1 mM MgCl2, 1,000 mM NaCl.

      The Gα protein should be eluted with 20% of Q buffer B.

    12. The FPLC program for target Gα elution is shown in Figure 3 and the Gα was eluted out as shown in the graph, in which the flow rate, maximum column pressure, and fraction were set to 1.0 ml/min, 0.5 MPa, and 2.0 ml, respectively.



      Figure 3. Purification of yeast-derived Gα proteins. A. SDS-PAGE for the fractions after his-tag purification. B. The elution program and resultant elution profile for Gα. C. SDS-PAGE for concentrated fraction from FPLC marked in (B).


    13. The corresponding fractions were collected and concentrated to 1 ml using Ultra-15 Centrifugal filters 3K at 4 °C with the speed of 3,846 × g.

    14. The concentration of the Gα was measured using BCA kit with a productivity of 2-5 mg/L cell culture.


    Purification of Gβγ
    1. Cell pellets were harvested by centrifugation at 3,800 × g for 10 min using the centrifuge at 4 °C in 250 ml centrifuge bottles. The supernatant was discarded.

    2. Cell pellets were washed once with Common Washing Buffer P1 in the ratio of 1 g cell pellets to 2 ml P1 and centrifuge at 3,800 × g for 10 min at 4 °C in 250 ml centrifuge bottles.

    3. Cell pellets were washed one time with 1:2 ratio Common Washing Buffer P1 and centrifuged at 4 °C with the speed of 3,800 × g for 10 min.

    4. The cell pellets were suspended in ice-cold G Protein Lysis Buffer P2.

    5. The cell pellets were lysed by Microfluidizer for 4 cycles at a pressure of 15,000 psi. The Microfluidizer was balanced with Buffer P2 prior to lysis.

    6. The lysed cell pellets were centrifuged at 9,720 × g for 30 min to remove all debris and intact cells in 250 ml centrifuge bottles.

    7. The supernatant was applied to Talon resin for 2 h.

      Note: Imidazole was added to the final concentration of 100 μM in order to decrease non-specific binding proteins.

    8. The G protein bound resins were applied onto a disposal column.

    9. The column was washed with 5 column volumes of Common Washing buffer P1.

    10. The Gβγ was eluted with 10 ml G Protein Elution Buffer at a gravity rate. Strategically recycling the elution buffer to reduce the elution volume was optional.

    11. The Gβγ was concentrated to 2 ml by Ultra-15 Centrifugal filters 3K at 4 °C with the speed of 3,846 × g. and changed the buffer into Q Sepharose High performance Buffer A-Gβγ.

    12. The sample was applied onto the FPLC and eluted with gradient elution with Q Sepharose High performance Buffer B-Gβγ (Figure 4A).

    13. Fractions were collected using the same parameters for the Gα; a typical elution profile was as follows (Figure 4):



      Figure 4. Purification of yeast-derived Gβγ proteins. A. The elution program and resultant elution profile for Gβγ. B. SDS-PAGE for the concentrated fractions marked in (A).


    14. The eluted fractions were collected and concentrated with a productivity of 2-5 mg/L cell culture similar to Gα. The purity and molecular weight of proteins were validated using SDS-PAGE as shown in Figure 4B.

Recipes

  1. 10× Phosphate buffer

    49.7 g Na2HPO4

    98.0 g NaH2PO4 in 1,000 ml dH2O, pH 6.5

  2. Immunoblotting Blocking buffer

    125 mM NaCl

    25 mM Tris base, pH 7.5, 0.3% Tween-20 and 3% non-fat milk

  3. Immunoblotting Incubation buffer

    Anti-His/Anti-Flag antibody diluted to 1:2,000 with blocking buffer

  4. Immunoblotting Washing buffer

    125 mM NaCl

    25 mM Tris base, pH 7.5

    0.3% Tween-20

  5. LB plates

    2.5% LB broth

    1.5% Agar

  6. YNBD plates

    1.34% Yeast Nitrogen based w/o amino acid

    0.0004% D-Biotin

    1% Dextrose

    1.5% Agar

  7. YPD medium

    1% Yeast extract

    2% Peptone

    2% Dextrose

  8. YPD agar plates

    1% Yeast extract

    2% Peptone

    2% Glucose

    2% Agar

  9. BMGY medium

    1% (w/v) Yeast extract

    2% (w/v) Peptone

    1.34% (w/v) YNB without amino acids

    0.00004% (w/v) Biotin

    1% (w/v) Glycerol

    0.1 M Phosphate buffer at pH 6.5

  10. BMMY medium

    1% (w/v) Yeast extract

    2% (w/v) Peptone

    1.34% (w/v) Yeast nitrogen base without amino acids

    0.00004% (w/v) Biotin

    0.5% (w/v) Methanol

    0.1 M Phosphate buffer at pH 6.5

    0.04% (w/v) Histidine and 3% (v/v) DMSO

    10 μM Theophylline

  11. Common Washing Buffer P1

    20 mM Bis-Tris, pH 6.5 (50 mM HEPES, pH 7.4)

  12. Receptor Lysis Buffer P2

    20 mM Bis-Tris, pH 6.5 (50 mM HEPES, pH 7.4)

    2.5 mM EDTA

    10% Glycerol

  13. Receptor Preparation Buffer P3

    20 mM Bis-Tris, pH 6.5 (50 mM HEPES, pH 7.4)

    100 mM NaCl

    1% MNG-3 and 0.02% CHS

  14. Receptor Preparation Column Washing Buffer P4

    20 mM Bis-Tris, pH 6.5 (50 mM HEPES, pH 7.4)

    100 mM NaCl

    0.1% MNG-3 and 0.002% CHS

  15. Column Elution Buffer P5

    20 mM Bis-tris, pH 6.5 (50 mM HEPES, pH 7.4)

    100 mM NaCl

    0.1% MNG-3

    0.002% CHS

    300 mM Imidazole

  16. XAC Column Elution Buffer P6

    20 mM theophylline in Receptor Receptor Preparation Column Washing Buffer P4

  17. G Protein Washing Buffer P1

    50 mM HEPES, pH 8.0

  18. G Protein Lysis buffer P2

    50 mM HEPES, pH 8.0

    10% Glycerol

    100 mM NaCl

  19. G Protein Elution Buffer P3

    50 mM HEPES, pH 8.0, 300 mM imidazole

  20. Q Buffer A- Gα

    50 mM HEPES, pH 8.0

    50 μM GDP

    1 mM MgCl2

  21. Q Buffer B- Gα

    50 mM HEPES, pH 8.0

    50 μM GDP

    1 mM MgCl2

    1,000 mM NaCl

  22. Q Buffer A-Gβγ

    20 mM Bis-Tris, pH 6.5 (50 mM HEPES, pH 8.0)

  23. Q Buffer B-Gβγ

    20 mM Bis-Tris, pH 6.5 (50 mM HEPES, pH 8.0)

    1,000 mM NaCl

  24. Immunoblotting Blocking Buffer

    125 mM NaCl

    25 mM Tris base, pH 7.5

    0.3% Tween-20

    3% Non-fat milk

  25. Immunoblotting Washing Buffer

    125 mM NaCl

    25 mM Tris base, pH 7.5

    0.3% Tween-20

  26. Radioligand Binding/Washing Buffers

    25 mM HEPES, pH 7.4

    100 mM NaCl

Acknowledgments

The article was supported by Nexus Initiative (L. Y.) from University of South Florida (USF) as well as startup funding (L.Y) from the department of Cell Biology, Microbiology and Molecular Biology at USF.

Competing interests

There are no conflicts of interest or competing interest.

References

  1. Andre, N., Cherouati, N., Prual, C., Steffan, T., Zeder-Lutz, G., Magnin, T., Pattus, F., Michel, H., Wagner, R. and Reinhart, C. (2006). Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen. Protein Sci 15(5): 1115-1126.
  2. Congreve, M., de Graaf, C., Swain, N. A. and Tate, C. G. (2020). Impact of GPCR Structures on Drug Discovery. Cell 181(1): 81-91.
  3. Dubnau, D. (1999). DNA uptake in bacteria. Annu Rev Microbiol 53: 217-244.
  4. Gallagher, S. and Chakavarti, D. (2008). Immunoblot analysis. J Vis Exp (16). doi:10.3791/759.
  5. Krettler, C., Reinhart, C. and Bevans, C. G. (2013). Expression of GPCRs in Pichia pastoris for structural studies. Methods Enzymol 520: 1-29.
  6. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T. Nature 227(5259): 680-685.
  7. Overington, J. P., Al-Lazikani, B. and Hopkins, A. L. (2006). How many drug targets are there. Nat Rev Drug Discov 5(12): 993-996.
  8. Smbrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning: a laboratory manual. Cold Spring harbor laboratory.
  9. Ye, L., Neale, C., Sljoka, A., Lyda, B., Pichugin, D., Tsuchimura, N., Larda, S. T., Pomes, R., Garcia, A. E., Ernst, O. P., Sunahara, R. K. and Prosser, R. S. (2018). Mechanistic insights into allosteric regulation of the A2A adenosine G protein-coupled receptor by physiological cations. Nat Commun 9(1): 1372.
  10. Ye, L., Orazietti, A. P., Pandey, A. and Prosser, R. S. (2018). High-Efficiency Expression of Yeast-Derived G-Protein Coupled Receptors and 19F Labeling for Dynamical Studies. Methods Mol Biol 1688: 407-421.
  11. Ye, L., Van Eps, N., Zimmer, M., Ernst, O. P. and Prosser, R. S. (2016). Activation of the A2A adenosine G-protein-coupled receptor by conformational selection. Nature 533(7602): 265-268.

简介

[摘要]在过去的几年中,作为GPCR-G复杂结构数量激增的证据,用于结构生物学的GPCR和G蛋白的表达已取得了巨大的成功,主要是在昆虫和哺乳动物细胞系统中,导致了370多个已解决了70多个GPCR的结构。但是,挑战仍然存在,特别是在构象转变和动力学研究领域,即使与X射线和冷冻EM相比(5 mg / ml,3μl /样品),也需要大量的受体和G蛋白。当应用NMR光谱法(5 mg / ml,250μl /样品)时。结果,i的表达水平 nsect和哺乳动物系统也很难满足这一需求,更不用说使用绝大多数系统使用这些系统生产GPCR和G蛋白的成本高昂了。因此,需要探索一种具有广泛适用性的有效,负担得起的实用方法。毕赤酵母表达系统已在GPCR制备中显示出其希望,并具有其他真核表达系统无法比拟的许多优点。在该系统中表达的GPCR价格便宜,易于操作,并且能够进行同位素标记。在此,我们提出最近开发并在我们的实验室升级的相关协议,包括表达和纯化的毕赤酵母衍生GPCR以G沿α和G βγ蛋白。我们预期这些协议将促进GPCR及其复合物的构象转变和动力学研究。


[背景] G蛋白偶联受体(GPCR)是最大的膜蛋白家族,在许多(病理)生理活动中起着关键作用。GPCR的或它们的效应物的功能障碍会导致各种病症,包括神经变性疾病,癌症,和慢性炎症(Overington等人,2006) 。虽然超过350层的结构(康格里夫等人,2020 )ø ˚F超过70 GPCRs在复杂的解决与配体和/或transducins ,我们对如何理解的信令通过受体在配体结合到下游信号传播伙伴仍难以捉摸,部分是由于解析的结构仅反映了这些复合物的动态信号传递过程的静态快照。此外,结构本身的局限性包括柔性域的缺失部分以及许多残留物的侧链信息,而这些残留物在X射线和冷冻-EM光谱学中均无法解析。发展互补性的办法来获取丢失的信息将提供宝贵的见解,拓宽GPCR的连续信号处理的我们的理解和引导我们的药物发现目标具体malfunctional信号。

显然,NMR是一种极好的工具,可在不影响静态结构的情况下,以最小的结构功能扰动来询问GPCR及其信号伴侣的构象转变和动力学。在NMR研究中,通常使用野生型或修饰程度最低的构建体,当在模拟生理环境(例如洗涤剂MNG-3,胶束或高密度脂蛋白颗粒)中进行操作时,可以最大程度地反映受体的真实行为(重建后的HDL)系统。NMR研究的特定要求也提出了常规表达系统(如大肠杆菌异源表达系统)的挑战。尽管这是最简单,最有效的系统,但表达的蛋白质(尤其是人类衍生的蛋白质)缺乏翻译后修饰s ,这通常会导致功能丧失。插件ECT细胞如SF9,SF21,或HI5,和哺乳动物表达系统,如HEK293和CHO细胞越来越多地用于结构生物学特别是对于X射线和冷冻电镜分光螺柱IES其中的受体相对少量的小号是需要。然而,用于NMR研究,需要一个大的蛋白质quantit ý沿着同位素标记使得昆虫和哺乳动物系统中挑战第要采用的类型为研究。因此,我们开发了一种基于酵母的表达系统来产生受体,其转导蛋白可以满足这一要求。在这个协议中,我们特别专注于制备GPCR的一个广泛适用的策略,G α和G βγ ,其中,用于GPCR制备该协议进一步升级基于我们以前的出版物(叶等人,2016,2018A和2018B )有显著提高了生产率。对于g α和G βγ制剂已被纳入本组协议作为用于GPCR-G蛋白复合物的研究的完整体系。在手稿中描述的策略也可应用到其他的信令伙伴如GRKs和β -抑制蛋白。如果需要,也很容易切换到同位素标记。与上述其他系统相比,在巴斯德毕赤酵母系统中表达这些受体和效应子的优势是显而易见的。

关键字:G蛋白偶联受体, G蛋白, 酵母, 构造, 动力学, 快速蛋白液相色谱, 高收益的生产力

材料和试剂
1.5 ml微量离心管(Fisher Scientific,目录号:05-408-130)
2 mm电穿孔比色皿(Fisher Scientific,目录号:FB102)
硝酸纤维素膜(Bio- R ad,目录号:1620112)
Nalgene TM Rapid- Flow TM带有尼龙膜的一次性过滤器灭菌装置(Thermo Scientific,目录号:09-740-24A)
Fisherbrand TM散装HDPE 7 ml闪烁瓶(Fisher Scientific,目录号:03-337-20)
Whatman TM无粘结剂玻璃微纤维过滤器,GF / C级(Cytiva ,目录号:09-874-32A)
DWK Life Sciences Kimble TM固体硼硅酸盐玻璃珠(Fisher S cientific,目录号:10-310-3)
清教徒TM医院标准棉签(费舍尔小号系统求解,目录号:22-029-684)
Sartoris TM Vivaspin TM 20离心浓缩器(Sartorius,目录号:14-558-501)
热科学TM的Nalgene TM速效流TM灭菌一次性过滤单元与尼龙膜(Fisher Scientific公司,目录号:0974024A)
滑动-A- Lyzer TM MINI透析装置,3.5K MWCO,0.5毫升(费舍尔小号系统求解,目录号:88400)
14 ml圆底管(Fisher Scientific,目录号:12-565-971)
250 ml的的Nalgene ®离心瓶(Sigma-Aldrich公司,目录号:Z353736)
巴斯德毕赤酵母SMD 1163菌株(Invitrogen)
pPIC9K载体(Invitrogen)
pPIC9K_ADORA2A(由pPIC9K设计)
XL-10- Gold TM超能感受态大肠杆菌细胞(安捷伦,目录号:200314)
BCA蛋白测定试剂盒(Pierce,目录号:PI23227)
咪唑(Fisher Chemical edge,产品目录号:03196-500)
EDTA(Sigma-Aldrich,目录号:E5134)
冰醋酸(Fisher Scientific,目录号:A385-500)
甲醇(Fisher Scientific,目录号:412-500或A412P4)
甘油(Fisher Scientific,目录号:G33-500)
d山梨糖醇(费希尔小号系统求解,目录号:S459)
G418硫酸盐(费舍尔小号系统求解,目录号:BP6735或野牛之公司,目录号:400-130-XG)
氨苄西林钠盐(Wisent Inc.,目录号:400-110-EG或Fisher Fisher,目录号:BP176025)
LB米勒汤(Wisent Inc,目录号:800-061-1K)
LB琼脂米勒粉末(费舍尔小号系统求解,目录号:BP14252)
酵母提取物(Wisent Inc.,目录号:800-150-IK)
细菌学第一肽(Wisent Inc.,目录号:800-157-IK)
酵母氮源W / O阿明ø酸(野牛之公司,目录号:800152018WG)
右旋糖(d葡萄糖)(费希尔小号系统求解,目录号:D16-500)
琼脂(费希尔小号系统求解,目录号:BP1423-500)
D-生物素(Sigma-Aldrich,目录号:2031)
L-组氨酸(Sigma-Aldrich公司,目录号:H8000-5G或Fisher小号系统求解,目录号:BP382100)
DMSO(Fisher Scientific,目录号:D1391)
茶碱(西格玛奥德里奇,目录号:T1633)
的NaCl(费希尔小号系统求解,目录号:S271-500)
双- Ť RIS(费舍尔小号系统求解,目录号:BP301-500)
十二烷基麦芽糖新戊二醇(MNG-3)(Anatrace ,目录号MG310)
胆固醇半琥珀酸酯(CHS)(Sigma-Aldrich,目录号:C6512)
Talon金属亲和树脂(TaKaRa ,目录号:635503)
(可选)液氮
Bam HI -HF(New England BioLabs ,目录号:R3136S)
无TI -HF(新英格兰生物实验室,目录号:R3189S)
PM EI (新英格兰生物实验室,目录号:R0560S)
T4 DNA连接酶(新英格兰生物实验室,目录号:M0202S)
快速CIP(新英格兰生物实验室,目录号:M0525S)
GenElute TM质粒微量制备试剂盒(Sigma-Aldrich,目录号:PLD35-1KT)
100%乙醇(Fisher Scientific,目录号:A4094)
HRP缀合的6 *他,His-标签单克隆抗体(Proteintech ,目录号:HRP-66005)
DDDDK TAG多克隆抗体(相当于FLAG ®抗体)(Proteintech ,目录号:HRP-20543-1-AP)
Zymolyase -20T(Amsbio ,目录号:120493-1)
黄嘌呤胺同类物(Sigma-Aldrich,目录号:X103)
Affi-Gel TM 10(Bio- R广告,目录号:1536099)
异丙醇(费希尔小号系统求解,目录号:A426P-4)
消泡剂A浓缩液(Sigma-Aldrich, 货号: A5633)
Thermo Scientific TM酵母DNA提取试剂盒(Fisher Scientific,目录号:PI78870)
Thermo Scientific TM Y-PER TM酵母蛋白质提取试剂(Fisher Scientific,目录号:PI78991)
Universal-ES液体闪烁鸡尾酒(MP Biomedicals TM ,目录号:88248001)
3,3',5,5'-四甲基联苯胺溶液(Alfa Aesar ,目录号:J61325)
PageRuler加上预染的蛋白梯(费舍尔小号系统求解,目录号:26619)
BioAcryl -P(30%,29:1)液体(Alfa Aesar ,目录号:J63279)
QIAquick凝胶提取试剂盒(Qiagen,目录号:28704)
10 ×磷酸盐缓冲液(请参见配方)
免疫印迹阻断缓冲液(请参见配方)
免疫印迹孵育缓冲液(请参阅食谱)
免疫印迹洗涤缓冲液(请参见配方)
LB板(请参阅食谱)
YNBD板(请参阅食谱)
YPD介质(请参阅食谱)
YPD琼脂平板(请参阅食谱)
BMGY培养基(请参见食谱)
BMMY培养基(请参阅食谱)
通用洗涤缓冲液P1(请参阅配方)
受体裂解缓冲液P2(请参阅食谱)
受体制备缓冲液P3(请参阅食谱)
受体制备柱洗涤缓冲液P4(请参阅食谱)
色谱柱洗脱缓冲液P5(请参阅配方)
XAC柱洗脱缓冲液P6(请参阅配方)
G蛋白洗涤缓冲液P1(请参阅食谱)
G蛋白裂解缓冲液P2(请参阅食谱)
G蛋白洗脱缓冲液P3(请参阅食谱)
Q缓冲液A-Gα(请参阅配方)
Q缓冲液B-Gα(请参阅食谱)
Q缓冲液AGβγ (请参阅食谱)
Q缓冲液BGβγ (请参阅食谱)
免疫印迹封闭缓冲液(请参见配方)
免疫印迹洗涤缓冲液(请参见食谱)
放射性配体结合/洗涤缓冲液(请参见食谱)

设备


(可选)-80°C冰箱
热科学TM SORVALL TM离心机(Thermo Scientific的,米Odel等:ST 40R)
Eppendorf TM微量离心机(Eppendorf,型号:Legend Micro 21R)
Applied Biosystems TM SimpliAmp TM热循环仪(Applied Biosystems,型号:A24812)
Genesys TM紫外可见光谱仪(Genesys ,模式:10S)
热科学TM Heratherm微生物培育器117L(Thermo Scientific的,米Odel等:IGS100)
NanoDrop TM分光光度计(NanoDrop ,型号:Lite)
贝克曼L8-70M超速离心机(贝克曼,型号:L8-70M)
ÄKTApurifierFPLC (GE,型号:Purifier 100)
细胞电穿孔系统电穿孔仪(Bio-Rad,型号:Gene Pulser X)
FISHERBRAND TM重型Vortex混合器(FISHERBRAND ,米Odel等:STD)
5 L NEW不伦瑞克生物反应器(新不伦瑞克,米Odel等:BIOFLO III)
微流化TM处理器(IDEX材料加工,米Odel等:LM20)

程序


该协议已根据我们之前的工作(Ye等人,2016和2018b )以及当前工作进行了修改和最终确定。与我们以前的工作不同,使用来自IDT网站的在线密码子优化软件对该协议中提出的所有靶基因进行了密码子优化,并分别合成了各个基因,然后通过Bam HI和Not HI限制酶整合到巴斯德毕赤酵母的基因组中。特别是,人源腺苷A 2A R(35.1KDa)和A 1受体(36.5KDa)基因在N和C末端分别具有FLAG和His 10标签。此外,在FLAG标签之后,将TEV蛋白酶切割位点以及α-因子肽添加到基因的前面(图1)。在此相反,没有FLAG标签和α -因子序列G中α (45.7KDa),以及为G β (38.7KDa)和G ^ γ (7.6KDa)构建体; 特别是,没有他的标签序列摹γ考虑摹β和g ^ γ共表达在一起。





图1.本方案中描述的GPCR和G蛋白的构建体。该质粒pPIC9K-A 1个R / pPIC9K中-A 2A - [R具有α -因子肽,FLAG标签,TEV蛋白酶切割。相反,在G蛋白构建体中没有FLAG标签和α-因子序列。特别是,没有他的标签在主题摹γ考虑摹β和g ^ γ共表达在一起。


GPCR和G蛋白的质粒制备
1个 μ升的construc的吨的pP我Ç 9K_ADORA2A这是pPIC9K中的重组载体含有ADORA2A受体序列,在〜100的浓度纳克/微升,由T.小林(日本)慷慨提供(安德烈等人。,2006 )用化学方法转化成2微升通过热激45秒,在42 XL-10金感受态细胞℃。

200 μ升LB培养基立即加入到混合物中,并转化细胞都在含有5025毫升LB平板扩散 μ克/米升氨苄青霉素。在37 °C下孵育过夜。
ø NE菌落被挑选从板和接种d成一个含有4毫升LB培养基50  μ克/米升氨苄青霉素和培养d在37过夜℃下。
按照说明使用GenElute TM质粒微型制备试剂盒提取质粒。
所提取的质粒用1消化μ升的Bam HI -HF AND1 μ升 无tI -HF用1 × NEB CutSmart ®缓冲˚F或在1.5ml Eppendorf管中1.5小时。1 μ升快速CIP是为30分钟的消化和磷酸加入到创建pPIC9K中骨干轴承的Bam HI和无TI在每一侧上的限制性位点。总体积随所用质粒的量而变化。一般,1微升的每个限制性酶是用于20的消化μ克质粒。
所有的靶基因片段,包括甲2A R,A 1 R,G α小号,G β ,和G ^ γ (〜200毫微克/ μ升)按照合成巴斯德毕赤酵母密码子优化的序列。值得注意的是,所有基因片段在N和C末端都带有Bam HI和No tI限制酶位点。
的基因片段,也消化1 μ升每个的Bam HI -HF和无TI -HF限制性内切酶用1 × NEB CutSmart ®缓冲器,用于无急CIP处理2小时。
消化的pPIC9K质粒和基因片段均经过DNA电泳(Krettler等,2013 ),并使用QIAquick凝胶提取试剂盒纯化,并使用NanoDrop TM Lite分光光度计测量浓度。   
将连接处理用骨架质粒和基因片段之间各种比例使用1处理微升T4 DNA连接酶与1 ×在0.2毫升PCR管NEB T4 DNA连接酶缓冲液。Ť他摩尔比基因之间fragements和空质粒分别设定为1:3,1:1和3:1的10-100纳克/浓度μ升 。
含有不同基因片段连接后的质粒转化到XL-10金的感受态细胞(Smbrook等,1989; Dubnau ,1999 )和涂布在含有50所述LB平板 μ克/米升氨苄青霉素。在37 °C下孵育过夜。 
使用GenElute TM Plasmid Miniprep Kit提取每种构建体的质粒。20 μ克质粒在100每个构建体的μl用1线性化 微升经Pme我限制用1酶× NEB CutSmart ®缓冲器,用于在一个0.2毫升PCR管2小时。
200 μ升100%乙醇加入到100 μ升的线性化质粒和在冰上温育5分钟。
然后将线性化的质粒在4°C下以16,200 × g的速度离心5分钟。
弃去上清液,并将线性化的DNA沉淀干燥UND ER一个烟气罩20分钟前重新悬浮在20 μ升的蒸馏水与DNA浓度约为1 μ克/ μ升。
10 μ升的线性化质粒的用80轻轻混合μ升巴斯德毕赤酵母感受态细胞在1.5ml Eppendorf管中并在冰上保持至前5分钟转移到一个2mm电比色皿。
的转化,使用基因脉冲II电穿孔用的1的条件下进行,500伏电压充电,25 μF的电容,并且400Ω的电阻。
立即将1 ml冰冷的1 M山梨糖醇加入电穿孔比色杯中,并转移到14 ml圆底试管中。
然后将样品在30 °C下温育3小时,不摇动,然后将其铺展到YNBD板上。
值得注意的是,线性化的G ^ β和g ^ γ质粒(10 μ升每个),我们重新转化到巴斯德毕赤酵母一起,而所有其他的线性化的质粒分别转化。

GPCR和G蛋白高产构建体的制备
如图2A所示,在YNBD平板上生长菌落后3-5天的潜伏期被转移到含有YPD平板1mg / ml的G418和我ncubate d 3-5天,在30 ℃下。制备了梯度G418浓度的YPD平板,并保存在4 °C进行后续筛选。





图2.电穿孔和高产率构建体筛选腺苷A 2A R和A 1 R受体的示意图(A)以及使用SDS-PAGE和Western印迹法进行鉴定(B)


将在含有1 mg / ml G418的YPD平板上生长的菌落进一步转移到含有2 mg / ml G418的第二个YPD平板上。在30 °C下孵育3-5天。
连续地,最终将在含有2mg / ml G418的YPD平板上生长的菌落转移到含有4mg / ml G418的YPD平板上。孵育ð额外3-5天,在30 ℃下。
5-10菌落在含有YPD平板4毫克/毫升G418分别使用针对免疫印迹测定法采摘表达水平评估他10个FLAG标签。pPIC9K中的多拷贝整合可以增加遗传霉素® 0.5阻力一级毫克/毫升(12份)到4毫克/毫升(7 - 12个拷贝)。
将单一菌落在摇动(275 rpm)下于30 °C的14 ml F alcon管中的4 ml BMGY培养基中接种至少24 h,直到OD 595 = 2.0-6.0。
然后将培养基转移入200ml BMMY培养基在500ml摇所涵盖瓶中一个棉塞。
将细胞继续在22 ℃下以275rpm摇动培养60小时。
将0.5%甲醇/ 12 h加入培养基中,以诱导和维持受体或G蛋白的表达。
在诱导结束时,将细胞沉淀物离心分离后收集在3 ,800 ×克10分钟在4 ℃下在250ml离心瓶中。
细胞沉淀用普通洗涤缓冲液P1以1:2的比例洗涤一次。
然后以等于1:4的细胞沉淀与缓冲液之比将细胞沉淀重悬浮于受体裂解缓冲液P2中。
然后将细胞沉淀悬浮液在5 mm玻璃珠浆液的存在下于2,000 rpm在4 °C涡旋2 h 。
将破碎的细胞沉淀物在4°C下以9,720 × g离心30分钟,并丢弃未破碎的细胞和细胞碎片。
收集含有细胞膜的上清液并进行免疫印迹分析(Gallagher和Chakavarti ,2008 )。
为了准确起见,分别针对FLAG标签和His标签对抗His和抗FLAG进行了免疫印迹。
将1μl上清液印迹在硝酸纤维素膜上并使其干燥。
将第二个1μl的上清液加到先前的位置,使其干燥。
将膜在室温下置于20 ml免疫印迹封闭缓冲液中1小时。
然后将膜转移至20ml我mmunoblotting孵育缓冲液含有任一His标签抗体(1:2 ,000)或FLAG -tag抗体(1:2 ,000)2小时。
然后将膜用免疫印迹洗涤缓冲液洗涤3次,然后用蒸馏水洗涤。
膜最终通过BM Blue POD底物(3,3',5,5'-四甲基联苯胺溶液)可视化。
选择强度最强的菌落用于进一步表达。如果表达水平少于0.5毫克/大号细胞培养物,第二循环的质粒转化中的指令之后进行在Pro描述cedure甲和B.菌落直接筛选含6毫克/毫升G418的YPD平板上(图URE 2A)。
用20%高压灭菌的甘油制备经筛选的高产率表达构建体的储备液,并在-80°C的冰箱或液氮中冷冻。

GPCR和G蛋白的表达
GPCR和G蛋白的表达使用以下步骤中类似的方法,但有细微的差异。

将转化子的甘油储备液接种到含有0.1 mg / ml G418的YPD琼脂平板上。
3-5天后,将单个菌落接种到14 ml圆底管中,在30°C下于4 ml高压灭菌的YPD中,以275 rpm振摇24 h。
随后将4 ml培养基转移到200 ml高压灭菌器中 BMGY培养基,在500 ml摇瓶中于30°C培养 覆盖由铝箔275的转速,直到OD的振荡速度595达到2-6,这将需要约24小时。
为了诱导表达,将200ml细胞沉淀物离心分离并转移至2.8L烧瓶中的1升高压灭菌的BMMY培养基中,并在20℃以275rpm的振荡速度培养。过滤甲醇溶液每12小时(5毫升/添加有0.5%大号配有鎓)如果折流使用烧瓶。如果发酵罐是所使用的,甲醇的加入速率以最大为1ml / h的控制每个升,这是依赖的培养物体积上
发酵80小时后收集受体表达细胞,而60小时后收集G蛋白表达细胞。

GPCR和G蛋白的纯化
的GPCR,G的纯化α ,和G βγ蛋白质是在这个协议不同。因此,我们在以下各节中分别描述它们。


GPCR的纯化

表达80小时后,将细胞沉淀物于3800 × g于4 °C在250 ml离心瓶中收集10分钟。
细胞沉淀物用普通洗涤缓冲液P1以1 g细胞沉淀物与2 ml P1的比率洗涤一次,并在3800 × g下于4 °C在250 ml离心瓶中离心10分钟。
将洗涤过的细胞沉淀以4:1的比例重悬于受体裂解缓冲液P2中,以确保在250 ml离心瓶中充分悬浮。
然后使用微流化器在15,000 psi的工作压力下在冰上将细胞破坏四个周期。裂解前,将微流化剂与缓冲液P2平衡。
我ntact细胞和细胞碎片从膜悬浮液在9720分离×克在4℃下30分钟,250 ml的 离心瓶中。
然后收集上清液,并使用带有相应试管的Beckman Ultracentrifuge的T ype 45 Ti转子以100,000 × g离心75分钟。
弃去超速离心的上清液,并收集不同运行的膜,并将其悬浮在通用洗涤缓冲液P1中以除去EDTA。
弃去超速离心的上清液,将膜沉淀溶解在装有受体制备缓冲液P3的50 ml锥形离心管中,并在4°C摇动直至所有膜充分溶解。
将该溶液在4°C下以1,980 × g离心5分钟以除去未溶解的膜。
使用受体制备柱洗涤缓冲液P4将溶解的膜与预平衡的Talon树脂混合。通常,将2 ml Talon树脂用于6 g膜。在4°C下孵育2小时。在孵育过程中,添加咪唑至终浓度为100μM 。
2小时后,将Talon树脂填充到处理柱上,并用5倍柱体积的受体制备柱洗涤缓冲液P4洗涤。
随后,使用柱洗脱缓冲液P5以重力从柱上洗脱受体。
洗脱的受体浓缩至1 ml 通过Ultra-15离心过滤器3K和10 ml受体制备柱洗涤缓冲液P4进行一次更换,在4 °C下以10的稀释倍数以10 3846 ×克。
然后,受体通过XAC配体柱,该XAC配体柱使用Receptor Preparation Column Washing Buffer P4进行了预平衡。重复三遍。
使用受体制备柱洗涤缓冲液P4洗涤结合了XAC受体的XAC色谱柱,体积为2倍,以去除非功能性受体。
然后使用XAC柱洗脱缓冲液P6洗脱受体,该缓冲液P6由具有25 mM茶碱的受体制备柱洗涤缓冲液P4组成。
洗脱的受体通过3K Ultra-15离心过滤器浓缩至1 ml,并用SlideK -A- Lyzer TM MINI透析设备3.5K对受体制备柱洗涤缓冲液P4透析,稀释因子为10 6,以除去所有配体,从而使受体进入载脂蛋白状态。
将纯化的受体通常与0.5-2毫克/大号生产率通过SDS-PAGE验证(的Laemmli ,1970 )作为WEL升免疫印迹,如图2B所示。

的G纯化α

细胞小球中收获由离心在3 ,800 ×克为10分钟使用的在4离心机在250ml离心瓶中℃下。
细胞沉淀物用一次洗涤共洗涤缓冲液P1中的比例为1克细胞沉淀至2ml P1和具有3800的速度离心×克为10分钟,在4在250ml离心瓶中℃下。
将细胞沉淀以1:4的比例重悬于G蛋白裂解缓冲液P2中。
通过Microfluidizer在15,000 psi下使用4个循环破碎细胞,以完全破坏酵母细胞壁。裂解前,将微流化剂与缓冲液P2平衡。
将裂解液在250毫升离心瓶中于4°C离心30分钟,离心9,720 ×g 。
将上清液施加到达隆树脂2小时,在将其用100的终浓度加入咪唑μ中号,以便降低非特异性结合。
将结合了G蛋白的Talon树脂应用于处理柱。
然后用5倍柱体积的G蛋白洗涤缓冲液P1洗涤填充柱。
用10 ml G蛋白洗脱缓冲液P3以重力洗脱目标Gα。从战略RECYCL荷兰国际集团洗脱缓冲液,以减少洗脱体积是可选的。
毫克氯2加入至1mM的最终浓度以及在50 GDP μ中号。
的Gα通过超15离心过滤器浓缩至2ml,3K在4 ℃下以3的速度,846 ×克。在将洗脱的Gα蛋白应用到系统之前,将FPLC系统用Q Sepharose色谱柱的缓冲液A-Gα平衡。
Q缓冲器A-Gα:50mM的HEPES,pH值8.0,50 μ中号GDP,1毫摩尔MgCl 2 。
Q缓冲器B-Gα:50mM的HEPES,pH值8.0,50 μ中号GDP,1毫摩尔MgCl 2 ,1000毫米的NaCl 。
应使用20%的Q缓冲液B洗脱Gα蛋白。

为目标Gα洗脱FPLC程序示于图3和Gα被洗脱出来如该曲线图所示,在该流率,最大列压力,和级分设定为1.0毫升/分钟,0.5兆帕,和2.0ml的, 分别。



图3.酵母来源的Gα蛋白的纯化。一。His-tag纯化后的馏分的SDS-PAGE。乙。洗脱程序和用于所得洗脱曲线G ^ α 。Ç 。SDS-PAGE用于(B)中标记的来自FPLC的浓缩级分。


对应的级分收集,浓缩至1使用超15离心过滤器3K以4ml ℃下与3的速度,846 ×克。
使用BCA试剂盒测量Gα的浓度,生产率为2-5 mg / L细胞培养。

Gβγ的纯化

        细胞小球中收获由离心在3800 ×克为10分钟使用的离心机在4在250ml离心瓶中℃下。 弃去上清液。
        将细胞沉淀物以1 g细胞沉淀物与2 ml P1的比率用Common Washing Buffer P1洗涤一次,并在3800 x g的条件下于4°C在250 ml离心瓶中离心10分钟。
        Ç ELL佩尔Ë TS瓦特ERE洗涤一次以1:2的比例共洗涤缓冲液P1,并在4离心℃下的速度3800 ×克为10分钟。
        将细胞沉淀物悬浮在冰冷的G蛋白裂解缓冲液P2中。
        将细胞球粒进行溶解通过微射流均质机为4个周期在一个压力的为15,000psi。裂解前,将微流化剂与缓冲液P2平衡。 
        Ť ħ Ë升ý SED Ç ELL佩尔Ë TS分别为Ç Ë Ñ三˚F uged一吨9720 ×克为30分钟吨ö ř EM ö已经所有德b RIS一个Ñ d我Ñ圆通Ç厄尔在250ml离心瓶中。
        将上清液施加到Talon树脂上2小时。
注意:添加咪唑至终浓度为100μM ,以减少非特异性结合蛋白。

        将结合G蛋白的树脂施加到处理柱上。
        用5倍柱体积的通用洗涤缓冲液P1洗涤该柱。
    对于g βγ在重力速率用10毫升聚体G蛋白的洗脱缓冲液洗脱。从战略RECYCL荷兰国际集团洗脱缓冲液,以减少洗脱体积是可选的。
    对于g βγ物通过超离心15浓缩至2ml过滤3K在4 ℃下与3846的速度×克。 并将缓冲液改为Q Sepharose高性能缓冲液AGβγ 。
    将样品上样到FPLC上,并用Q Sepharose高性能缓冲液BGβγ梯度洗脱(图4A)。
    级分用用于G的相同参数收集α ; 典型的洗脱曲线如下(图4):



图4.酵母来源的Gβγ蛋白的纯化。一。洗脱程序和用于所得洗脱曲线ģ βγ 。乙。SDS-PAGE用于(A)中标记的浓缩级分。


    洗脱的级分收集,浓缩,之后用2-5毫克/的生产率大号类似于G细胞培养α 。使用SDS-PAGE验证蛋白质的纯度和分子量,如图4B所示。

菜谱


10 ×磷酸盐缓冲液
49.7克Na 2 HPO 4

98.0克的NaH 2 PO 4在1 ,000毫升卫生署2 O,pH 6.5的

免疫印迹阻断缓冲液
125毫米氯化钠

25 mM Tris碱,pH 7.5,0.3%Tween-20和3%脱脂牛奶

免疫印迹孵育缓冲液
抗-His /抗-Flag 2:稀释至1抗体,000用封闭缓冲液

免疫印迹洗涤缓冲液
125毫米氯化钠

25 mM Tris碱,pH 7.5

0.3%吐温20

LB板
2.5%LB肉汤

1.5%琼脂

YNBD板
1.34%不含氮的酵母氮

0.0004%D-生物素

1%葡萄糖

1.5%琼脂

YPD培养基
1%酵母提取物

2%蛋白ept

2%葡萄糖

YPD琼脂板
1%酵母提取物

2%蛋白ept

2%葡萄糖

2%琼脂

BMGY培养基
1%(w / v)酵母提取物

2%(w / v)蛋白ept

1.34%(w / v)YNB,不含氨基酸

0.00004%(w / v)生物素

1%(w / v)甘油

pH 6.5的0.1 M磷酸盐缓冲液

BMMY培养基
1%(w / v)酵母提取物

2%(w / v)蛋白ept

1.34%(w / v)不含氨基酸的酵母氮碱

0.00004%(w / v)生物素

0.5%(w / v)甲醇

pH 6.5的0.1 M磷酸盐缓冲液

0.04%(w / v)组氨酸和3%(v / v)DMSO

10 μM茶碱

通用洗涤缓冲液P1
20 mM Bis- T ris,pH 6.5(50 mM HEPES,pH 7.4)

受体裂解缓冲液P2
20 mM Bis- T ris,pH 6.5(50 mM HEPES,pH 7.4)

2.5毫米EDTA

10%甘油

受体制备缓冲液P3
20 mM Bis- T ris,pH 6.5(50 mM HEPES,pH 7.4)

100毫米氯化钠

1%MNG-3和0.02%CHS

受体制备柱洗涤缓冲液P4
20 mM Bis- T ris,pH 6.5(50 mM HEPES,pH 7.4)

100毫米氯化钠

0.1%MNG-3和0.002%CHS

柱洗脱缓冲液P5
20 mM Bis-tris,pH 6.5(50 mM HEPES,pH 7.4)

100毫米氯化钠

0.1%MNG-3

0.002%CHS

300 mM咪唑

XAC柱洗脱缓冲液P6
受体制备柱洗涤缓冲液P4中20 mM茶碱

G蛋白洗涤缓冲液P1
50 mM HEPES,pH 8.0

G蛋白裂解缓冲液P2
50 mM HEPES,pH 8.0

10%甘油

100毫米氯化钠

G蛋白洗脱缓冲液P3
50 mM HEPES,pH 8.0,300 mM咪唑

Q缓冲液A-Gα
50 mM HEPES,pH 8.0

50 μ中号GDP

1毫米氯化镁2

Q缓冲液B-Gα
50 mM HEPES,pH 8.0

50 μ中号GDP

1毫米氯化镁2

1 ,000毫氯化钠

Q缓冲A-摹βγ
20 mM Bis- T ris,pH 6.5(50 mM HEPES,pH 8.0 )

Q缓冲B-摹βγ
20 mM Bis- T ris,pH 6.5(50 mM HEPES,pH 8.0 )

1 ,000毫氯化钠

免疫印迹封闭缓冲液
125毫米氯化钠

25 mM Tris碱,pH 7.5

0.3%吐温20

3%脱脂牛奶

免疫印迹洗涤缓冲液
125毫米氯化钠

25 mM Tris碱,pH 7.5

0.3%吐温20

放射性配体结合/洗涤缓冲液
25 mM HEPES,pH 7.4

100毫米氯化钠


致谢


该文章得到了南佛罗里达大学(USF)的Nexus Initiative(LY)以及USF细胞生物学,微生物学和分子生物学系的启动资金(LY)的支持。


利益争夺


没有利益冲突或利益冲突。


参考


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叶,L.,尼尔,C.,Sljoka ,A.,LYDA ,B.,皮丘金,D.,Tsuchimura ,N.,Larda ,ST,梨果,R.,加西亚,AE,恩斯特,OP,砂原,RK和Prosser,RS(2018 a )。机械洞察力通过生理阳离子对A2A腺苷G蛋白偶联受体的变构调节。Nat Commun 9(1):1372。
Ye,L.,Orazietti,AP,Pandey,A.和Prosser,RS(2018 b )。酵母衍生的G蛋白偶联受体的高效表达和19 F标记用于动力学研究。方法分子生物学1688:407-421。
Ye,L.,Van Eps,N.,Zimmer,M.,Ernst,OP和Prosser,RS(2016)。通过构象选择激活A2A腺苷G蛋白偶联受体。自然533(7602):265-268。
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引用:Zhao, W., Wang, X. and Ye, L. (2021). Expression and Purification of Yeast-derived GPCR, Gα and Gβγ Subunits for Structural and Dynamic Studies. Bio-protocol 11(4): e3919. DOI: 10.21769/BioProtoc.3919.
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