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Producing GST-Cbx7 Fusion Proteins from Escherichia coli
利用大肠埃希杆菌制备GST-Cbx7融合蛋白   

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Abstract

This protocol describes the production of GST-Cbx7 fusion proteins from E. coli, originally developed in the recent publication (Zhen et al., 2016). The pGEX-6P-1-GST plasmids encoding the Cbx7 variants were transformed into BL21 competent cells. The fusion protein production was induced by isopropyl-beta-D-thiogalactopyranoside and they were purified by Glutathione Sepharose 4B. This protocol can be adapted for the purification of other proteins.

Keywords: Polycomb(多梳), Cbx7(Cbx7), GST-fusion proteins(GST融合蛋白), pGEX-6P-1(pGEX-6P-1), Affinity purification(亲和纯化), Glutathione Sepharose 4B(谷胱甘肽琼脂糖4B)

Background

Polycomb group (PcG) proteins regulate gene expression by modulating higher order chromatin structures (Kerppola, 2009; Simon and Kingston, 2013). PcG proteins are generally found in two major complexes, Polycomb repressive complex (PRC) 1 and 2 (Kerppola, 2009; Simon and Kingston, 2013). PRC2 is a methyltransferase that catalyzes di- and tri-methylation of lysine 27 on histone H3 (H3K27me2/3) (Cao et al., 2002); PRC1 is an ubiquitin ligase that monoubiquitylates histone H2A on lysine 119 (H2AK119Ub) (Wang et al., 2004). Mammalian PRC1 complexes are further divided to canonical and variant PRC1 (Gao et al., 2012, Tavares et al., 2012). Canonical PRC1 is composed of one of each Ring1 (Ring1A/Ring1B), Pcgf (Mel18/Bmi1), Phc (Phc1/2/3), and Cbx (Cbx2/4/6/7/8) proteins. The Cbx family proteins have a conserved chromodomain (CD) that recognizes H3K27me3, suggesting molecular links between the recruitment of canonical PRC1 and H3K27me3 (Blackledge et al., 2015). Recently, we have interrogated the molecular mechanisms underlying the binding of Cbx7-PRC1 to chromatin by live-cell single-molecule imaging (Zhen et al., 2016). We showed that the CD and AT-hook-like (ATL) motif of Cbx7 constitute a functional DNA-binding unit by electrophoretic mobility shift assay (Zhen et al., 2016). Here, detailed conditions are presented which allow the production of GST-Cbx7 fusion proteins from E. coli. With modifications, this protocol may be used for the purification of other proteins. The purification of fusion proteins by GST fusion system has been widely applied in various biochemical and structural studies (Harper and Speicher, 2011).

Materials and Reagents

  1. 50 ml conical tube
  2. Dialysis membrane (Spectra/Por® Molecularporous membrane tubing, standard RC tubing, MWCO: 3.5 kD) (Spectrum, catalog number: 132720 )
  3. Bio-Rad Chromatography Column (2.5 x 10 cm Econo-Column) (Bio-Rad Laboratories, catalog number: 7311550 )
  4. BL21 competent cells (made in the laboratory)
  5. pGEX-6P-1-GST (GE Healthcare)
  6. Ampicillin
  7. Isopropyl-1-thio-β-D-galactopyranoside (IPTG) (Omega Bio-tek, catalog number: AC121 )
  8. Glutathione Sepharose 4B (GE Healthcare, catalog number: 17075601 )
  9. Phosphate-buffered saline (PBS, pH 7.4)
  10. PierceTM Coomassie (Bradford) Protein Assay Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23200 )
  11. Coomassie Blue (Bio-Rad Laboratories, catalog number: 1610786 )
  12. Tryptone
  13. Yeast extract
  14. Sodium chloride (NaCl)
  15. 1% Triton X-100
  16. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 93482 )
  17. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 )
  18. L-glutathione reduced (Sigma-Aldrich, catalog number: G4251 )
  19. LB medium (see Recipes)
  20. Wash buffer (see Recipes)
  21. Lysis buffer (see Recipes)
  22. Elution buffer (see Recipes)

Equipment

  1. Culture flasks (2,000 ml) (NALGENE, U.S.A.)
  2. Shaker
  3. Centrifuges (Eppendorf, model: 5702 R )
  4. Centrifuge bottle
  5. Vibra-CellTM sonicator (Sonics & Materials, model: VCX 130 ) with standard probe (1/4”; 6 mm), length 4.5” (113 mm), Titanium alloy Ti-6Al-4V, Autoclavable (Sonics & Materials, catalog number: 630-0435 )
  6. S-3200-2 GyroMixer (BioExpress, model: GeneMate GyroMixer , catalog number: S-3200-2)

Procedure

Note: The experimental procedure was revised from the published protocol (Harper and Speicher, 2011).

  1. Expression of GST fusion protein
    1. Transform competent BL21 cells with pGEX-6P-1-GST plasmids that encode the Cbx7 variants by incubating plasmid with cells on ice for 10 min, then heat shock at 42 °C for 45 sec. The mixture was put on ice for 2 min, LB medium was added, and the mixture was incubated for 1 h at 37 °C while shaking at 250-300 rpm. After that, the cells were spread onto agar plate containing ampicillin.
    2. Transfer a single, isolated colony of transformed BL21 cells to 100 ml LB medium with 100 µg/ml ampicillin and incubate the inoculated culture overnight at 37 °C while shaking at 250-300 rpm.
    3. Transfer 50 ml of the overnight culture into 950 ml of warm, fresh LB medium with 100 µg/ml ampicillin.
    4. Incubate the culture at 37 °C while shaking at 250-300 rpm until the OD600 is 0.5-0.7 (Note 1).
    5. Induce the protein expression by adding IPTG to a final concentration of 1.0 mM (stock concentration: 100 mM, the powder was dissolved in MilliQ water and the solution was filtered before used) and incubating at 37 °C while shaking at 250-300 rpm for 5 h.
    6. Harvest cells by centrifugation at 4,000 x g for 20 min at 4 °C.
    7. Carefully decant the supernatant, leaving about 15-50 ml in the centrifuge bottle.
    8. Resuspend the cells and transfer to a 50 ml conical tube and centrifuge at 4,000 x g for 20 min at 4 °C.
    9. Decant the supernatant (Note 2).

  2. Sonication (Note 3)
    1. Resuspend the cell pellet in 25 ml of lysis buffer.
    2. Lyse cells by sonication at 4 °C using the standard probe with the following settings:
      1. 15-sec on
      2. 45-sec off
      3. 45% input (45% amplitude)
      4. 6 min: total time on

  3. Purification
    1. To the mixture after sonication, add Triton X-100 to a final concentration of 1% and mix gently for 30 min at 4 °C with the mixer to increase the solubility of the protein.
    2. Centrifuge the mixture at 10,000 x g for 10 min at 4 °C.
    3. Wash 0.75 ml of Glutathione Sepharose beads with 3 x 10 ml of cold PBS, centrifuge at 500 x g for 3 min at 4 °C.
    4. Transfer the supernatant by pipetting from step C2 to pre-washed beads in a conical tube and rotate at 4 °C for 1 h.
    5. Centrifuge at 500 x g for 3 min at 4 °C and remove the supernatant.
    6. Wash the beads with 4 x 10 ml of wash buffer.
    7. Pour the beads into a Bio-Rad Chromatography Column and wash with 2 x 10 ml of cold PBS.
    8. Elute the fusion protein by incubating at room temperature for 10 min with 1 ml elution buffer. Repeat this step to get a total of three elutions.
    9. Dialyze against PBS three times.
    10. Run SDS-PAGE gel to determine the purity and identity of the fusion protein (Figure 1).


      Figure 1. SDS-PAGE gel stained with Coomassie for determination of Cbx7 variant GST-fusion proteins

Data analysis

The PierceTM Coomassie (Bradford) Protein Assay Kit (Thermo Scientific) was performed to determine the concentration of the protein. SDS-PAGE gel was used to determine the identity of the proteins by their expected molar mass and also to check for contaminates (if yes, there will be other bands shown together with the protein band). Furthermore, GST protein was run with the SDS-PAGE gel as a control. The gel was stained with Coomassie Blue and is presented in Figure 6-figure supplement 1 in (Zhen et al., 2016): Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin.

Notes

  1. To monitor the OD, measure the absorbance at 600 nm. Estimate the amount of time by assuming the population of E. coli doubles every 20 min.
  2. The cell pellet from step C10 can be frozen at -80 °C for several months.
  3. For sonication: cell disruption is evidenced by partial clearing of the suspension. Avoid over sonication since it will heat the solution, leading to protein aggregation and denaturation.
  4. After sonication, 1% Triton X-100 was added, Triton X-100 can be replaced with other detergents such as NP-40 or Tween 20.

Recipes

  1. LB medium (Autoclaved, 1 L, pH 7.2)
    10 g tryptone
    5 g yeast extract
    5 g NaCl
  2. Wash buffer
    1x PBS + 1% Triton X-100
  3. Lysis buffer
    1x PBS
    0.1 mM PMSF
    0.1 mM protease inhibitor cocktail
  4. Elution buffer
    20 mM reduced glutathione, pH 8.0
    Adjust pH with NaOH

Acknowledgments

This work was supported, in whole or in part, by the National Cancer Institute of the National Institutes of Health under Award Number R03CA191443 (to XR). This work was also supported by grants from the CU-Denver Office Research Service (to XR) and the American Cancer Society Grant IRG 57-001-53 subaward (to XR).
This protocol was modified from (Harper and Speicher, 2011).

References

  1. Blackledge, N. P., Rose, N. R. and Klose, R. J. (2015). Targeting Polycomb systems to regulate gene expression: modifications to a complex story. Nat Rev Mol Cell Biol 16(11): 643-649.
  2. Cao, R., Wang, L., Wang, H., Xia, L., Erdjument-Bromage, H., Tempst, P., Jones, R. S. and Zhang, Y. (2002). Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298(5595): 1039-1043.
  3. Gao, Z., Zhang, J., Bonasio, R., Strino, F., Sawai, A., Parisi, F., Kluger, Y. and Reinberg, D. (2012). PCGF homologs, CBX proteins, and RYBP define functionally distinct PRC1 family complexes. Mol Cell 45(3): 344-356.
  4. Harper, S. and Speicher, D. W. (2011). Purification of proteins fused to glutathione S-transferase. Methods Mol Biol 681: 259-280.
  5. Kerppola, T. K. (2009). Polycomb group complexes--many combinations, many functions. Trends Cell Biol 19(12): 692-704.
  6. Simon, J. A. and Kingston, R. E. (2013). Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put. Mol Cell 49(5): 808-824.
  7. Tavares, L., Dimitrova, E., Oxley, D., Webster, J., Poot, R., Demmers, J., Bezstarosti, K., Taylor, S., Ura, H., Koide, H., Wutz, A., Vidal, M., Elderkin, S. and Brockdorff, N. (2012). RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3. Cell 148(4): 664-678.
  8. Wang, H., Wang, L., Erdjument-Bromage, H., Vidal, M., Tempst, P., Jones, R. S. and Zhang, Y. (2004). Role of histone H2A ubiquitination in Polycomb silencing. Nature 431(7010): 873-878.
  9. Zhen, C. Y., Tatavosian, R., Huynh, T. N., Duc, H. N., Das, R., Kokotovic, M., Grimm, J. B., Lavis, L. D., Lee, J., Mejia, F. J., Li, Y., Yao, T. and Ren, X. (2016). Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin. Elife 5.

简介

该方案描述了最近在最近的出版物(Zhen等,2016)中开发的大肠杆菌GST-Cbx7融合蛋白的生产。 将编码Cbx7变体的pGEX-6P-1-GST质粒转化到BL21感受态细胞中。 通过异丙基-β-D-硫代吡喃半乳糖苷诱导融合蛋白的产生,并用谷胱甘肽琼脂糖4B纯化。 该方案可以适用于其他蛋白质的纯化。
【背景】Polycomb组(PcG)蛋白通过调节高阶染色质结构调节基因表达(Kerppola,2009; Simon和Kingston,2013)。 PcG蛋白通常存在于两种主要复合物Polycomb镇压复合物(PRC)1和2(Kerppola,2009; Simon和Kingston,2013)中。 PRC2是甲基转移酶,其催化组蛋白H3(H3K27me2 / 3)上赖氨酸27的二甲基和三甲基化(Cao等,2002); PRC1是在赖氨酸119(H2AK119Ub)上单核苷酸组氨酸H2A的泛素连接酶(Wang等,2004)。哺乳动物PRC1复合物进一步分为典型和变体PRC1(Gao等,2012,Tavares等,2012)。规范PRC1由每个Ring1(Ring1A / Ring1B),Pcgf(Mel18 / Bmi1),Phc(Phc1 / 2/3)和Cbx(Cbx2 / 4/6/7/8)蛋白之一组成。 Cbx家族蛋白具有保守的染色质结构域(CD),其识别H3K27me3,表明在标准PRC1和H3K27me3的招募之间的分子联系(Blackledge等人,2015)。最近,我们已经询问了Cbx7-PRC1通过活细胞单分子成像与染色质结合的分子机制(Zhen等,2016)。我们显示Cbx7的CD和AT钩状(ATL)基序通过电泳迁移率变动测定构成功能性DNA结合单元(Zhen等,2016)。这里提供了允许从大肠杆菌生产GST-Cbx7融合蛋白的详细条件。通过修改,该方案可用于纯化其它蛋白质。 GST融合系统融合蛋白的纯化已广泛应用于各种生化和结构研究(Harper and Speicher,2011)。

关键字:多梳, Cbx7, GST融合蛋白, pGEX-6P-1, 亲和纯化, 谷胱甘肽琼脂糖4B

材料和试剂

  1. 50ml圆锥管
  2. 透析膜(Spectra/Por 分子膜管,标准RC管,MWCO:3.5kD)(Spectrum,目录号:132720)
  3. Bio-Rad色谱柱(2.5×10cm经济柱)(Bio-Rad Laboratories,目录号:7311550)
  4. BL21感受态细胞(实验室制造)
  5. pGEX-6P-1-GST(GE Healthcare)
  6. 氨苄青霉素
  7. 异丙基-1-硫代-β-D-吡喃半乳糖苷(IPTG)(Omega Bio-tek,目录号:AC121)
  8. 谷胱甘肽琼脂糖4B(GE Healthcare,目录号:17075601)
  9. 磷酸盐缓冲盐水(PBS,pH 7.4)
  10. Pierce TM 考马斯(Bradford)蛋白测定试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,目录号:23200)
  11. 考马斯蓝(Bio-Rad Laboratories,目录号:1610786)
  12. 胰蛋白胨
  13. 酵母提取物
  14. 氯化钠(NaCl)
  15. 1%Triton X-100
  16. 苯基甲磺酰氟(PMSF)(Sigma-Aldrich,目录号:93482)
  17. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8340)
  18. L-谷胱甘肽还原(Sigma-Aldrich,目录号:G4251)
  19. LB培养基(参见食谱)
  20. 洗涤缓冲液(见配方)
  21. 裂解缓冲液(见配方)
  22. 洗脱缓冲液(见配方)

设备

  1. 培养瓶(2,000ml)(NALGENE,U.S.A。)
  2. 振动器
  3. 离心机(Eppendorf,型号:5702 R)
  4. 离心瓶
  5. 具有标准探针(1/4"; 6mm),长度4.5"(113mm),钛合金Ti-6Al-的超声波超声仪(Sonics& Materials,型号:VCX 130) 4V,Autoclavable(Sonics& Materials,catalog number:630-0435)
  6. S-3200-2 GyroMixer(BioExpress,型号:GeneMate GyroMixer,目录号:S-3200-2)

程序

注意:实验程序从已发布的协议(Harper和Speicher,2011)进行了修订。

  1. GST融合蛋白的表达
    1. 通过将质粒与细胞在冰上孵育10分钟,然后在42℃下热休克45秒,通过用编码Cbx7变体的pGEX-6P-1-GST质粒转化感受态的BL21细胞。将混合物放在冰上2分钟,加入LB培养基,并将混合物在37℃下孵育1小时,同时以250-300rpm摇动。之后,将细胞铺在含有氨苄青霉素的琼脂平板上。
    2. 将单个分离的转化的BL21细胞的菌落转移到具有100μg/ml氨苄青霉素的100ml LB培养基中,并在37℃下孵育接种培养物一夜,同时以250-300rpm摇动。
    3. 将50ml过夜培养物转移到具有100μg/ml氨苄青霉素的950ml温热的新鲜LB培养基中
    4. 在37℃下孵育培养物,同时以250-300rpm振荡直至OD 600为0.5-0.7(注1)。
    5. 通过加入IPTG至最终浓度为1.0mM(储备浓度:100mM,将粉末溶解于MilliQ水中,溶液在使用前过滤)诱导蛋白质表达,并在37℃下以250-300rpm振荡孵育5 h。
    6. 在4℃下以4000g离心收获细胞20分钟。
    7. 小心地倒出上清液,在离心瓶中留下约15-50ml
    8. 重悬细胞并转移至50ml锥形管中,并在4℃下以4,000 x g离心20分钟。
    9. 倾倒上清液(注2)
  2. 超声处理(注3)
    1. 将细胞沉淀重悬于25ml裂解缓冲液中
    2. 使用标准探针在4°C下超声处理裂殖细胞,具有以下设置:
      1. 15秒在
      2. 45秒关闭
      3. 45%输入(45%幅度)
      4. 6分钟:总时间在

  3. 纯化
    1. 超声处理后,加入Triton X-100至终浓度1%,并在4℃下用混合器轻轻混合30分钟,以增加蛋白质的溶解度。
    2. 在4℃下以10,000×g离心混合物10分钟。
    3. 用3×10ml冷PBS洗涤0.75ml谷胱甘肽琼脂糖珠,在4℃下以500×g离心3分钟。
    4. 通过从步骤C2通过移液将上清液转移到锥形管中的预洗珠中,并在4℃下旋转1小时。
    5. 在4℃下以500×g离心3分钟,并除去上清液
    6. 用4×10ml洗涤缓冲液洗涤珠子。
    7. 将珠子倒入Bio-Rad色谱柱中,用2 x 10ml冷PBS洗涤
    8. 通过在室温下用1ml洗脱缓冲液孵育10分钟来洗脱融合蛋白。重复此步骤总共获得三个解决方案。
    9. 透析PBS三次。
    10. 运行SDS-PAGE凝胶以确定融合蛋白的纯度和同一性(图1)

      图1.用考马斯染色的用于测定Cbx7变体GST-融合蛋白的SDS-PAGE凝胶

数据分析

进行Pierce TM 考马斯(Bradford)蛋白质测定试剂盒(Thermo Scientific)测定蛋白质的浓度。使用SDS-PAGE凝胶通过其预期的摩尔质量来确定蛋白质的同一性,并检测污染物(如果是,将显示与蛋白质带一起显示的其它条带)。此外,用SDS-PAGE凝胶作为对照,运行GST蛋白。凝胶用考马斯蓝染色,并在图6的补充1中显示(Zhen等人,2016):活细胞单分子跟踪揭示了H3K27me3和DNA靶标的共识别多梳Cbx7-PRC1染色质。

笔记

  1. 为了监测OD,测量600 nm处的吸光度。假设大肠杆菌的人口每20分钟加倍一次估计时间。
  2. 来自步骤C10的细胞沉淀物可以在-80℃冷冻几个月。
  3. 超声处理:通过部分清除悬浮液证明细胞破裂。避免超声处理,因为它会加热溶液,导致蛋白质聚集和变性
  4. 超声处理后,加入1%Triton X-100,Triton X-100可用其他洗涤剂如NP-40或吐温20替代。

食谱

  1. LB培养基(高压灭菌,1升,pH 7.2) 10克胰蛋白胨
    5克酵母提取物
    5克NaCl
  2. 洗涤缓冲液
    1x PBS + 1%Triton X-100
  3. 裂解缓冲液
    1x PBS
    0.1 mM PMSF
    0.1mM蛋白酶抑制剂混合物
  4. 洗脱缓冲液
    20mM还原型谷胱甘肽,pH 8.0
    用NaOH调节pH值

致谢

这项工作全部或部分得到国家卫生研究院国家癌症研究所的奖励,编号为R03CA191443(至XR)。这项工作也得到了丹佛办事处研究处(XR)和美国癌症协会授予IRG 57-001-53向下(到XR)的资助。
该协议从(Harper和Speicher,2011)进行了修改。

参考

  1. Blackledge,NP,Rose,NR和Klose,RJ(2015)。  组蛋白H3赖氨酸27甲基化在Polycomb-group silencing中的作用。 科学 298(5595):1039-1043。
  2. Gao,Z.,Zhang,J.,Bonasio,R.,Strino,F.,Sawai,A.,Parisi,F.,Kluger,Y。和Reinberg,D。(2012)。< a class = PCGF同源物,CBX蛋白和RYBP定义了功能上不同的PRC1家族复合物的基因片段"ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22325352"。 Mol Cell 45(3):344-356。
  3. Harper,S.和Speicher,DW(2011)。  纯化与谷胱甘肽S-转移酶融合的蛋白质。方法Mol Biol 681:259-280。
  4. Kerppola,TK(2009)。 Polycomb组合 - 许多组合,许多功能。 Trends Cell Biol 19(12):692-704。
  5. Simon,JA和Kingston,RE(2013)。  占领染色质:用于获得基因组靶点的Polycomb机制,停止转录流量和保留。分子细胞 49(5):808-824。
  6. Tavares,L.,Dimitrova,E.,Oxley,D.,Webster,J.,Poot,R.,Demmers,J.,Bezstarosti,K.,Taylor,S.,Ura,H.,Koide, Wutz,A.,Vidal,M.,Elderkin,S.and Brockdorff,N。(2012)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22325148"target ="_ blank"> RYBP-PRC1复合物在独立于PRC2和H3K27me3的多聚靶位点上介导H2A泛素化。 148(4):664-678。 br />
  7. Wang,H.,Wang,L.,Erdjument-Bromage,H.,Vidal,M.,Tempst,P.,Jones,RS和Zhang,Y。(2004)。< a class ="ke-insertfile" href ="http://www.ncbi.nlm.nih.gov/pubmed/15386022"target ="_ blank">组蛋白H2A泛素化在Polycomb沉默中的作用自然 431( 7010):873-878。
  8. Zhen,CY,Tatavosian,R.,Huynh,TN,Duc,HN,Das,R.,Kokotovic,M.,Grimm,JB,Lavis,LD,Lee,J.,Mejia,FJ,Li,Y.,Yao ,T. and Ren,X.(2016)。  活细胞单分子跟踪显示H3K27me3和DNA靶标polycomb Cbx7-PRC1与染色质的共识识。 Elife 5.
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Copyright Huynh and Ren . This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Huynh, T. N. and Ren, X. (2017). Producing GST-Cbx7 Fusion Proteins from Escherichia coli. Bio-protocol 7(12): e2333. DOI: 10.21769/BioProtoc.2333.
  2. Zhen, C. Y., Tatavosian, R., Huynh, T. N., Duc, H. N., Das, R., Kokotovic, M., Grimm, J. B., Lavis, L. D., Lee, J., Mejia, F. J., Li, Y., Yao, T. and Ren, X. (2016). Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin. Elife 5.
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