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Signaling Assays for Detection of Human G-protein-coupled Receptors in Yeast
以酵母为模型对人G蛋白偶联受体进行信号传导分析   

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Abstract

G-protein-coupled receptors (GPCRs) are the largest group of cell-surface proteins and are major molecular targets for drug development. The protocol described herein is for the detection of human GPCR signaling in the yeast Saccharomyces cerevisiae. Using Zoanthus sp. green fluorescent protein (ZsGreen) as the reporter, engineered yeast cells expressing human GPCRs emit strong fluorescence in response to stimuli leading to receptor signal activation. This assay method would allow screening for agonistic ligands and critical mutations required for human GPCR signaling.

Keywords: Signaling assay(信号检测), G-protein-coupled receptor(G蛋白偶联受体), Biosensor(生物传感器), Yeast(酵母), Human GPCR(人类GPCR)

Materials and Reagents

  1. Engineered Saccharomyces cerevisiae strain in which the ZsGreen reporter genes were integrated into the genome (IMFD-72ZsD: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 sst2Δ::AUR1-C ste2Δ::LEU2 fig1Δ::ZsGreen his3Δ::PFIG1-ZsGreen far1Δ gpa1Δ::Gi3tp) (Nakamura et al., 2013)
    Note: The expression of ZsGreen is controlled by the signal-responsive FIG1 promoter.
  2. Multi-copy expression plasmid (pGK421 containing the PGK1 promoter, 2 μ origin and MET15 marker) (Togawa et al., 2010) encoding the GPCR of interest [e.g., somatostatin receptor subtype-5 (SSTR5); somatostatin receptor subtype-2 (SSTR2); or neurotensin receptor type-1 (NTSR1)] (Ishii et al., 2012; Ishii et al., 2014)
  3. GPCR ligands [e.g., somatostatin (SST) (Merck KGaA, Calbiochem®, catalog number: 51110-01-1 ) and neurotensin (NTS) (Merck KGaA, Calbiochem®, catalog number: 39379-15-2)]
  4. 10 mg/ml carrier DNA (Takara Bio Company, Clontech, catalog number: 630440 )
  5. Tris (hydroxymethyl) aminomethane (Tris.HCl) (Nacalai Tesque, catalog number: 35409-45 )
  6. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA 2Na∙2H2O) (Nacalai Tesque, catalog number: 15111-45 )
  7. 1 M Hydrochloric acid (HCl) (Nacalai Tesque, catalog number: 37314-15 )
  8. Lithium acetate dihydrate (Sigma-Aldrich, catalog number: L6883 )
  9. Acetic acid (Nacalai Tesque, catalog number: 00212-85 )
  10. Polyethylene glycol (PEG) #4000 (Nacalai Tesque, catalog number: 28221-05 )
  11. DMSO (Nacalai Tesque, catalog number: 13445-74 )
  12. BD FACSFlow sheath fluid (BD, catalog number: 342003 )
  13. Immersion oil (Olympus, catalog number: IMMOIL-F30CC )
  14. Distilled water (dH2O)
  15. Yeast extract (Nacalai Tesque, catalog number: 15838-45 )
  16. Peptone (BD, catalog number: 211677 )
  17. D-Glucose (Nacalai Tesque, catalog number: 16806-25 )
  18. Yeast nitrogen base without amino acids (YNB) (BD, catalog number: 291940 )
  19. L-Histidine (Nacalai Tesque, catalog number: 18116-92 )
  20. L-Leucine (Nacalai Tesque, catalog number: 20327-62 )
  21. Uracil (Nacalai Tesque, catalog number: 35824-82 )
  22. 3-(N-Morpholino)-2-hydroxypropanesulfonic acid (Mopso) (Nacalai Tesque, catalog number: 23421-64 )
  23. Sodium hydroxide (NaOH) (Nacalai Tesque, catalog number: 31511-05 )
  24. Agar (Nacalai Tesque, catalog number: 01028-85 )
  25. 10 x TE buffer (see Recipes)
  26. 10 x LiAc (see Recipes)
  27. 50% PEG (see Recipes)
  28. TE/LiAc solution (see Recipes)
  29. LiAc/PEG solution (see Recipes)
  30. YPD medium (see Recipes)
  31. Synthetic dextrose (SD) selective medium supplemented with histidine, leucine and uracil (see Recipes)
  32. SDM71 selective medium supplemented with histidine, leucine and uracil (see Recipes)

Equipment

  1. 16.5 x 105 mm test tubes (AGC Techno Glass, catalog number: 9820TST16.5-105NP )
  2. 10-ml conical flasks (AGC Techno Glass, catalog number: 4980FK10 )
  3. 2-ml microcentrifuge tubes (WATSON, catalog number: 332-720C )
  4. 1.5-ml microcentrifuge tubes (WATSON, catalog number: 131-815C )
  5. 96-well cell culture plate (Corning, catalog number: 3596 )
  6. Microplate sealing tape (AS ONE Corporation, catalog number: 1-6774-05 )
  7. 12.5 x 75 mm test tubes with 2-position caps (B & M Equipment, catalog number: 222-2036-050 )
  8. Microscope glass slides (Matsunami Glass, catalog number: S091150 )
  9. 18 x 18 mm coverslips (Matsunami Glass, catalog number: C218181 )
  10. Centrifuge (Eppendorf, model: MiniSpin plus )
  11. Block incubator (Astec Industries, model: BI-516C )
  12. Shaking incubator for test tubes and conical flasks (TAITEC, model: BR-43FL )
  13. Shaking incubator for a 96-well cell culture plate (TAITEC, model: M∙BR-022UP )
  14. Spectrophotometer (Shimadzu, model: UVmini-1240 )
  15. BD FACSCanto II flow cytometer (BD)
  16. Fluorescence microscope (Keyence Corporation, model: BZ-9000 )
  17. 100x objective lens (Nikon Corporation, model: CFI Plan Apo VC 100x H )
  18. BZ filter cube (excitation filter, absorption filter, dichroic mirror) (Keyence Corporation, model: OP-66836 GFP-BP )

Software

  1. BD FACSDiva software (v5.0) (BD)

Procedure

  1. Yeast transformation
    Transformation was performed using the lithium acetate method (Gietz et al., 1992).
    1. Inoculate 5 ml YPD medium in a 16.5 x 105 mm test tube with a single colony of S. cerevisiae strain IMFD-72ZsD.
    2. Incubate the culture overnight at 30 °C with shaking at 150 opm.
      Note: Shaking incubator (TAITEC, model: BR-43FL).
    3. Transfer 2 ml of the cell culture to a 2-ml microcentrifuge tube and pellet the cells by centrifugation at 3,000 rpm for 5 min at room temperature.
    4. Remove the supernatant and wash the cells with 1 ml sterilized dH2O.
    5. Pellet the cells by centrifugation at 3,000 rpm for 5 min at room temperature.
    6. Remove the supernatant and resuspend the cells in 1.5 ml of TE/LiAc solution.
    7. Aliquot 100 μl of yeast suspension to a fresh 1.5-ml microcentrifuge tube.
    8. Add 500 ng of plasmid DNA (pGK421-SSTR5, -SSTR2 or -NTSR1) and 20 μg of carrier DNA to each tube.
      Note: Before using the carrier DNA, denature it at 95 °C for 5 min, and then chill it quickly on ice.
    9. Add 600 μl of LiAc/PEG solution to each tube and vortex at high speed for 10 sec to mix.
    10. Incubate the cell suspension at 30 °C for 30 min.
    11. Add 70 μl of DMSO. Mix well by gentle inversion. Do not vortex.
    12. Heat shock at 42 °C for 15 min.
    13. Centrifuge the cell suspension at 14,000 rpm for 5 sec at room temperature. Remove the supernatant completely.
    14. Resuspend the cells in 0.5 ml of 1x TE buffer.
    15. Spread 100 μl of cell suspension on each SD selective agar plate.
      Note: Since the yeast strain has his3Δ leu2Δ met15Δ ura3Δ alleles and the plasmid has MET15 as a selection marker, the selective medium requires supplementation with histidine, leucine and uracil.
    16. Incubate the plates at 30 °C until colonies appear (generally, 2-4 days).

  2. GPCR signaling assay
    1. Inoculate 5 ml SD selective medium in a 16.5 x 105 mm test tube with a single colony of a positive transformant.
    2. Incubate the culture overnight at 30 °C with shaking at 150 opm.
      Note: Shaking incubator (TAITEC, model: BR-43FL).
    3. Measure the OD600 of the yeast cultures.
      Note: Spectrophotometer.
    4. Transfer the cultured cells into 5 ml of fresh SD selective medium in a 10-ml conical flask to give an initial OD600 of 0.03.
    5. Further incubate the culture at 30 °C for 18 h with rotary shaking at 150 rpm.
      Note: Shaking incubator (TAITEC, model: BR-43FL).
    6. Transfer 2 ml of the cell culture to a 2-ml microcentrifuge tube and pellet the cells by centrifugation at 3,000 rpm for 5 min at room temperature.
    7. Remove the supernatant and wash the cells with 1 ml sterilized dH2O.
    8. Pellet the cells by centrifugation at 3,000 rpm for 5 min at room temperature.
    9. Remove the supernatant and resuspend the cells in ~400 μl sterilized dH2O.
    10. Measure the OD600 of the cell suspension, and then dilute it to an OD600 of 10.
      Note: Spectrophotometer.
    11. Add 10 μl of the resulting cell suspension (to give an OD600 of 1) to 80 μl fresh SDM71 selective medium per well of a 96-well cluster dish.
    12. Add 10 μl of 10-times concentrated ligands (SST or NTS) or sterilized dH2O (no ligand control) to each well.
      Note: A final concentration of ~10 μM ligand is recommended (dissolved in and diluted with sterilized dH2O).
    13. Seal the 96-well cluster dish with sealing tape.
    14. Incubate the dish at 30 °C for 4 h with shaking at 150 rpm. After incubation, measure green fluorescence using a flow cytometer or fluorescence microscope (see below).
      Note: Shaking incubator (TAITEC, model: M∙BR-022UP).

      Flow cytometry analysis
      1. Dilute the cell culture in 1 ml sheath fluid in test tubes with 2-position caps.
      2. Measure the green fluorescence signal emitted from 10,000 cells using a BD FACSCanto II flow cytometer equipped with a 530/30 nm band-pass filter.
      3. Analyze the data and assess the mean ZsGreen fluorescence of 10,000 cells using BD FACSDiva software.

      Fluorescence microscopy imaging
      1. Transfer the cell culture to a 1.5-ml microcentrifuge tube and pellet the cells by centrifugation at 3,000 rpm for 5 min at room temperature.
      2. Remove the supernatant and wash the cells with 100 μl sterilized dH2O.
      3. Pellet the cells by centrifugation at 3,000 rpm for 5 min at room temperature.
      4. Remove the supernatant and resuspend the cells in 10 μl sterilized dH2O.
      5. Spot 7 μl of the cell suspension on a glass slide and mount a coverslip over the spot.
      6. Observe the cells using a BZ-9000 fluorescence microscope equipped with a 100x objective lens or equivalent and acquire green fluorescence images with a 470/40 band-pass filter for excitation and a 535/50 band-pass filter for emission.
        Note: The 100x objective lens should be immersed in oil.

Representative data



Figure 1. Activation of human SSTR5 produced in yeast following the exogenous addition of SST. Yeast strain IMFD-72ZsD was transformed with pGK421 (mock) or pGK421-SSTR5. All transformants were grown in SD medium for 18 h. The cells then were incubated for another 4 h in SDM71 medium with or without 10 μM SST. A. The ZsGreen fluorescence of 10,000 cells was measured by flow cytometry. Data are shown as means ± SDs of triplicate samples. B. Visualization of the ZsGreen fluorescence of IMFD-72ZsD/SSTR5 with or without 10 μM SST. Images were obtained using a 100x objective on a fluorescence microscope. Reprinted with permission from  Ishii et al. (2012).

Notes

  1. Work on a clean bench.

Recipes

  1. 10x TE buffer (200 ml)
    2.4 g Tris-HCl (0.1 M)
    0.74 g EDTA 2Na∙2H2O (10 mM)
    Dissolved in 175 ml dH2O
    Adjust pH to 7.5 with 1 M HCl
    Adjust volume to 200 ml with dH2O
    Sterilized by autoclaving
  2. 10x LiAc (200 ml)
    20.4 g lithium acetate dihydrate (1 M)
    Dissolved in 175 ml dH2O
    Adjust pH to 7.5 with dilute acetic acid
    Adjust volume to 200 ml with dH2O
    Sterilized by autoclaving
  3. 50% PEG (200 ml)
    100 g PEG #4000
    Add dH2O to 200 ml
    Sterilized by autoclaving
  4. TE/LiAc solution (1.5 ml)
    150 μl 10 x TE buffer
    150 μl 10 x LiAc
    1.2 ml sterilized dH2O
    Note: Prepare fresh just prior to use.
  5. LiAc/PEG solution (1 ml)
    800 μl 50% PEG
    100 μl 10 x TE buffer
    100 μl 10 x LiAc
    Note: Prepare fresh just prior to use.
  6. YPD medium (1 L)
    10 g yeast extract
    20 g peptone
    20 g D-Glucose
    Add dH2O to 1 L
    Sterilized by autoclaving
  7. SD selective medium supplemented with histidine, leucine and uracil (1 L)
    20 g D-Glucose
    6.7 g yeast nitrogen base without amino acids (YNB)
    20 mg L-Histidine
    60 mg L-Leucine
    20 mg Uracil
    Add dH2O to 1 L
    Sterilized by autoclaving
    Note: For solid plates, add 2% agar to the media.
  8. SDM71 selective medium supplemented with histidine, leucine and uracil (1 L)
    20 g D-Glucose
    1.7 g yeast nitrogen base without amino acids (YNB)
    45 g 3-(N-morpholino)-2-hydroxypropanesulfonic acid (Mopso)
    20 mg L-Histidine
    60 mg L-Leucine
    20 mg uracil
    Dissolved in 800 ml dH2O
    Adjust pH to 7.1 with 10 N NaOH
    Adjust volume to 1 L with dH2O
    Filter sterilized

Acknowledgments

This protocol was adapted from the previously published paper Nakamura et al. (2013). This work was supported by a Grant-in-Aid for JSPS Fellows (23∙2292) and for Young Scientists (B) (26820362) from the Japan Society for the Promotion of Science.

References

  1. Gietz, D., St Jean, A., Woods, R. A. and Schiestl, R. H. (1992). Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20(6): 1425.
  2. Ishii, J., Moriguchi, M., Hara, K. Y., Shibasaki, S., Fukuda, H. and Kondo, A. (2012). Improved identification of agonist-mediated Gαi-specific human G-protein-coupled receptor signaling in yeast cells by flow cytometry. Anal Biochem 426(2): 129-133.
  3. Ishii, J., Oda, A., Togawa, S., Fukao, A., Fujiwara, T., Ogino, C. and Kondo, A. (2014). Microbial fluorescence sensing for human neurotensin receptor type 1 using Gα-engineered yeast cells. Anal Biochem 446: 37-43.
  4. *Nakamura, Y., Ishii, J. and Kondo, A. (2013). Bright fluorescence monitoring system utilizing Zoanthus sp. green fluorescent protein (ZsGreen) for human G-protein-coupled receptor signaling in microbial yeast cells. PLos One 8(12): e82237.
  5. Togawa, S., Ishii, J., Ishikura, A., Tanaka, T., Ogino, C. and Kondo, A. (2010). Importance of asparagine residues at positions 13 and 26 on the amino-terminal domain of human somatostatin receptor subtype-5 in signalling. J Biochem 147(6): 867-873.

简介

G蛋白偶联受体(GPCR)是最大的细胞表面蛋白质组,是药物开发的主要分子靶标。 本文所述的方案用于在酵母酿酒酵母中检测人GPCR信号传导。 使用 Zoanthus 绿色荧光蛋白(ZsGreen )作为报告物,表达人GPCR的工程化酵母细胞响应于导致受体信号活化的刺激而发射强荧光。 该测定方法将允许筛选激动剂配体和人GPCR信号传导所需的关键突变。

关键字:信号检测, G蛋白偶联受体, 生物传感器, 酵母, 人类GPCR

材料和试剂

  1. 工程酿造酿酒酵母菌株,其中将ZsGreen 报道基因整合到基因组中(IMFD-72ZsD:MATahis3Δlleu2/em>Δem1 0 met15 0 ura3 0 sst2 Δ:: AUR1-C ste2 Δ :: Δgpa1Δ :: Gi3tp )(中村等人,2013)
    注意:ZsGreen的表达由信号响应性FIG1启动子控制。
  2. 将多拷贝表达质粒(含有PGK1启动子,2μ起点和 MET15 标记的pGK421)(Togawa等,编码感兴趣的GPCR(例如,生长抑素受体亚型-5(SSTR5));生长抑素受体亚型-2(SSTR2);或神经降压素受体I型(NTSR1)](Ishi等人,2012; Ishii等人,2014)。
  3. GPCR配体[例如,生长抑素(SST)(Merck KGaA,Calbiochem ,目录号:51110-01-1)和神经降压素(NTS)(Merck KGaA,Calbiochem& sup>®,目录号:39379-15-2)]
  4. 10mg/ml载体DNA(Takara Bio Company,Clontech,目录号:630440)
  5. 三(羟甲基)氨基甲烷(Tris缓冲盐酸)(Nacalai Tesque,目录号:35409-45)
  6. 乙二胺四乙酸二钠盐二水合物(EDTA 2Na·2H 2 O)(Nacalai Tesque,目录号:15111-45)
  7. 1M盐酸(HCl)(Nacalai Tesque,目录号:37314-15)
  8. 乙酸锂二水合物(Sigma-Aldrich,目录号:L6883)
  9. 乙酸(Nacalai Tesque,目录号:00212-85)
  10. 聚乙二醇(PEG)#4000(Nacalai Tesque,目录号:28221-05)
  11. DMSO(Nacalai Tesque,目录号:13445-74)
  12. BD FACSFlow鞘液(BD,目录号:342003)
  13. 浸没油(奥林巴斯,目录号:IMMOIL-F30CC)
  14. 蒸馏水(dH 2 O)
  15. 酵母提取物(Nacalai Tesque,目录号:15838-45)
  16. 蛋白胨(BD,目录号:211677)
  17. D-葡萄糖(Nacalai Tesque,目录号:16806-25)
  18. 无氨基酸的酵母氮源(YNB)(BD,目录号:291940)
  19. L-组氨酸(Nacalai Tesque,目录号:18116-92)
  20. L-亮氨酸(Nacalai Tesque,目录号:20327-62)
  21. 尿嘧啶(Nacalai Tesque,目录号:35824-82)
  22. 3 - (N,N-吗啉代)-2-羟基丙磺酸(Mopso)(Nacalai Tesque,目录号:23421-64)
  23. 氢氧化钠(NaOH)(Nacalai Tesque,目录号:31511-05)
  24. 琼脂(Nacalai Tesque,目录号:01028-85)
  25. 10 x TE缓冲区(参见配方)
  26. 10 x LiAc(参见配方)
  27. 50%PEG(参见配方)
  28. TE/LiAc溶液(参见配方)
  29. LiAc/PEG溶液(参见配方)
  30. YPD介质(参见配方)
  31. 补充有组氨酸,亮氨酸和尿嘧啶的合成葡萄糖(SD)选择性培养基(参见配方)
  32. 补充有组氨酸,亮氨酸和尿嘧啶的SDM71选择性培养基(参见Recipes)

设备

  1. 16.5×105mm试管(AGC Techno Glass,目录号:9820TST16.5-105NP)
  2. 10-ml锥形瓶(AGC Techno Glass,目录号:4980FK10)
  3. 2-ml微量离心管(WATSON,目录号:332-720℃)
  4. 1.5-ml微量离心管(WATSON,目录号:131-815C)
  5. 96孔细胞培养板(Corning,目录号:3596)
  6. 微孔板密封胶带(AS ONE Corporation,目录号:1-6774-05)
  7. 具有2位帽的12.5×75mm试管(B& M Equipment,目录号:222-2036-050)
  8. 显微镜载玻片(Matsunami Glass,目录号:S091150)
  9. 18×18mm盖玻片(Matsunami Glass,目录号:C218181)
  10. 离心机(Eppendorf,型号:MiniSpin plus)
  11. 块培养箱(Astec Industries,型号:BI-516C)
  12. 振荡用于试管和锥形瓶(TAITEC,型号:BR-43FL)的培养箱
  13. 摇动用于96孔细胞培养板(TAITEC,型号:M∙BR-022UP)的培养箱。
  14. 分光光度计(Shimadzu,型号:UVmini-1240)
  15. BD FACSCanto II流式细胞仪(BD)
  16. 荧光显微镜(Keyence公司,型号:BZ-9000)
  17. 100x物镜(尼康公司,型号:CFI Plan Apo VC 100x H)
  18. BZ滤波器立方体(激发滤波器,吸收滤波器,分色镜)(Keyence公司,型号:OP-66836GFP-BP)

软件

  1. BD FACSDiva软件(v5.0)(BD)

程序

  1. 酵母转化
    使用乙酸锂方法进行转化(Gietz等人,1992)。
    1. 在具有单菌落的16.5×105mm试管中接种5ml YPD培养基。 酿酒酵母菌株IMFD-72ZsD
    2. 将培养物在30℃,150℉条件下振荡培养过夜 注意:振荡培养箱(TAITEC,型号:BR-43FL)。
    3. 转移2毫升细胞培养物到2毫升微量离心管,并通过在室温下以3,000rpm离心5分钟沉淀细胞。
    4. 除去上清液并用1ml灭菌的dH 2 O洗涤细胞
    5. 通过在室温下以3,000rpm离心5分钟来沉淀细胞
    6. 除去上清液,并将细胞重悬于1.5ml TE/LiAc溶液中
    7. 等分100微升的酵母悬浮液到新鲜的1.5毫升微量离心管
    8. 向每个管中加入500ng质粒DNA(pGK421-SSTR5,-SSTR2或-NTSR1)和20μg载体DNA。
      注意:在使用载体DNA之前,在95℃变性5分钟,然后在冰上快速冷却。
    9. 向每个管中加入600μlLiAc/PEG溶液,并高速涡旋10秒以混合
    10. 在30℃下孵育细胞悬浮液30分钟
    11. 加入70μl的DMSO。 通过温和倒置混合。 不要涡旋。
    12. 在42℃热冲击15分钟。
    13. 在室温下以14,000rpm离心细胞悬浮液5秒。 完全除去上清液。
    14. 将细胞重悬于0.5ml 1×TE缓冲液中
    15. 在每个SD选择性琼脂平板上铺100微升细胞悬液 注意:由于酵母菌株具有his3Δleu2Δmet15Δura3Δ等位基因并且质粒具有MET15作为选择标记,因此选择培养基需要补充组氨酸,亮氨酸和尿嘧啶。
    16. 在30°C孵育平板,直到菌落出现(一般,2-4天)

  2. GPCR信号转导测定
    1. 在具有阳性转化体的单个菌落的16.5×105mm试管中接种5ml SD选择培养基。
    2. 将培养物在30℃,150℉条件下振荡培养过夜 注意:振荡培养箱(TAITEC,型号:BR-43FL)。
    3. 测量酵母培养物的OD <600> 注意:分光光度计
    4. 将培养的细胞转移到10ml新鲜的SD选择培养基中,在10ml锥形瓶中,初始OD600为0.03。
    5. 进一步在30℃下以150rpm的旋转振荡温育培养物18小时 注意:振荡培养箱(TAITEC,型号:BR-43FL)。
    6. 转移2毫升细胞培养物到2毫升微量离心管,并通过在室温下以3,000rpm离心5分钟沉淀细胞。
    7. 除去上清液并用1ml灭菌的dH 2 O洗涤细胞
    8. 通过在室温下以3,000rpm离心5分钟来沉淀细胞
    9. 除去上清液并将细胞重悬于〜400μl灭菌的dH 2 O中。
    10. 测量细胞悬浮液的OD 600,然后将其稀释至OD 600的10倍。
      注意:分光光度计。
    11. 将10μl所得细胞悬浮液(给予OD 600为1)加入到96孔簇培养皿的每孔80μl新鲜SDM71选择培养基中。
    12. 向每个孔中加入10μl10倍浓度的配体(SST或NTS)或灭菌的dH 2 O(无配体对照)。
      注意:建议最终浓度〜10μM配体(溶解于无菌dH 2 O中稀释)。< em>
    13. 用密封带密封96孔簇皿。
    14. 孵育皿在30℃下4小时,在150 rpm的摇动。孵育后,使用流式细胞仪或荧光显微镜(见下文)测量绿色荧光 注意:摇动培养箱(TAITEC,型号:M∙BR-022UP)。

      流式细胞术分析
      1. 稀释细胞培养在1毫升鞘液中的试管与2位帽
      2. 使用装备有530/30nm带通滤光片的BD FACSCanto II流式细胞仪测量从10,000个细胞发射的绿色荧光信号。
      3. 分析数据并使用BD FACSDiva软件评估10,000个细胞的平均ZsGreen荧光

      荧光显微镜成像
      1. 转移细胞培养物到1.5毫升微量离心管,并通过在室温下以3,000rpm离心5分钟沉淀细胞。
      2. 除去上清液,用100μl灭菌的dH 2 O洗涤细胞
      3. 通过在室温下以3,000rpm离心5分钟来沉淀细胞
      4. 除去上清液并将细胞重悬于10μl灭菌的dH 2 O中
      5. 点7玻璃载玻片上的细胞悬液,并在该点上盖上盖玻片
      6. 使用装备有100倍物镜或等效物的BZ-9000荧光显微镜观察细胞,并获得具有用于激发的470/40带通滤波器和用于发射的535/50带通滤波器的绿色荧光图像。
        注意:100x物镜应浸在油中。

代表数据



图1.外源性添加SST后在酵母中产生的人SSTR5的激活。用pGK421(mock)或pGK421-SSTR5转化酵母菌株IMFD-72ZsD。 所有转化体在SD培养基中生长18小时。 然后将细胞在具有或不具有10μMSST的SDM71培养基中孵育另外4小时。 A.通过流式细胞术测量10,000个细胞的ZsGreen荧光。 数据显示为一式三份样品的平均值±SD。 B.使用或不使用10μMSST的IMFD-72ZsD/SSTR5的ZsGreen荧光的可视化。 使用荧光显微镜上的100x物镜获得图像。 转载自Ishii 等人的许可。 (2012)。

笔记

  1. 在干净的长凳上工作。

食谱

  1. 10×TE缓冲液(200ml) 2.4克Tris-HCl(0.1M) 0.74g EDTA 2Na·2H 2 O(10mM) 溶于175ml dH 2 O中 用1M HCl调节pH至7.5 用dH 2 O调节体积至200ml
    高压灭菌
    灭菌
  2. 10x LiAc(200ml) 20.4g乙酸锂二水合物(1M) 溶于175ml dH 2 O中 用稀乙酸调节pH至7.5 用dH 2 O调节体积至200ml
    高压灭菌
    灭菌
  3. 50%PEG(200ml) 100g PEG#4000
    将dH 2 O加到200ml
    中 高压灭菌
    灭菌
  4. TE/LiAc溶液(1.5ml) 150μl10×TE缓冲液
    150μl10×LiAc
    1.2ml灭菌的dH 2 O
    注意:在使用前准备新鲜食物。
  5. LiAc/PEG溶液(1ml) 800μl50%PEG
    100μl10×TE缓冲液
    100μl10×LiAc
    注意:在使用前准备新鲜食物。
  6. YPD培养基(1L) 10g酵母提取物
    20克蛋白胨
    20克D-葡萄糖
    将dH <2> O添加到1 L
    高压灭菌
    灭菌
  7. 补充有组氨酸,亮氨酸和尿嘧啶(1L)的SD选择培养基 20克D-葡萄糖
    6.7g无氨基酸的酵母氮源(YNB)
    20mg L-组氨酸
    60 mg L-亮氨酸
    20mg尿嘧啶
    将dH <2> O添加到1 L
    高压灭菌
    灭菌 注意:对于实心板,向培养基中加入2%琼脂。
  8. 补充有组氨酸,亮氨酸和尿嘧啶(1L)的SDM71选择培养基 20克D-葡萄糖
    1.7g无氨基酸的酵母氮源(YNB)
    45g 3 - (N,N-吗啉代)-2-羟基丙磺酸(Mopso)
    20mg L-组氨酸
    60 mg L-亮氨酸
    20mg尿嘧啶
    溶于800ml dH 2 O中 用10N NaOH将pH调节至7.1 使用dH <2> O调节音量至1 L
    过滤灭菌

致谢

该协议改编自以前发表的论文Nakamura等人(2013)。这项工作得到了来自日本促进科学协会的JSPS研究员(23∙2292)和青年科学家(B)(26820362)的助学金的支持。

参考文献

  1. Gietz,D.,St Jean,A.,Woods,R.A。和Schiestl,R.H。(1992)。 改进的完整酵母细胞高效转化方法核酸研究 20(6):1425.
  2. Ishii,J.,Moriguchi,M.,Hara,K.Y.,Shibasaki,S.,Fukuda,H.and Kondo,A。(2012)。 改进了酵母细胞中激动剂介导的Gαi特异性人G蛋白偶联受体信号传导的鉴定流式细胞术。 Anal Biochem 426(2):129-133
  3. Ishii,J.,Oda,A.,Togawa,S.,Fukao,A.,Fujiwara,T.,Ogino,C.and Kondo,A。(2014)。 人类神经降压素受体的微生物荧光传感 1型使用Gα工程酵母细胞。 Anal Biochem 446:37-43。
  4. * Nakamura,Y.,Ishii,J.and Kondo,A。(2013)。 使用Zoanthus的明亮荧光监测系统 绿色荧光蛋白( Cy5)用于微生物酵母细胞中的人G蛋白偶联受体信号传导。 8(12):e82237。 >
  5. Togawa,S.,Ishii,J.,Ishikura,A.,Tanaka,T.,Ogino,C.and Kondo,A。(2010)。 人类生长抑素受体亚型的氨基末端结构域上第13和26位的天冬酰胺残基的重要性, 5在信号传导中。 147(6):867-873
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Nakamura, Y., Ishii, J. and Kondo, A. (2014). Signaling Assays for Detection of Human G-protein-coupled Receptors in Yeast. Bio-protocol 4(16): e1206. DOI: 10.21769/BioProtoc.1206.
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