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In vitro Protein Ubiquitination Assays
体外蛋白质泛素化修饰试验   

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Abstract

Ubiquitin can be added to substrate protein as a protein tag by the concerted actions of ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3). At the present of E1 and ubiquitin, E2 activity can be determined by the thio-ester formation. The E3 activity of a putative protein as well as the E2/E3 or E3/substrate specificities also can be explored by in vitro ubiquitination assay. The result can be detected by western blot with certain antibody. Purified proteins expressed from bacterial system are always used in this assay.

Keywords: Ubiquitination(泛素化), In vitro(体外), Ubiquitin ligase(泛素连接酶), Ubiquitin conjugating enzyme(泛素结合酶)

Materials and Reagents

  1. Cell crude extract
  2. Purified protein or crude extract of ubiquitin activating enzyme (E1)
  3. Purified recombinant ubiquitin conjugating enzyme (E2) fused with a protein tag (such as 6x His tag)
  4. Purified recombinant ubiquitin ligase (E3)
  5. Purified ubiquitin or recombinant ubiquitin protein fused with a tag ( (Kraft et al., 2005; Liu et al., 2010 )
  6. ATP (Sigma-Aldrich, catalog number: A7699 )
  7. MgCl2 (Sigma-Aldrich, catalog number: V900020 )
  8. Anti-ubiquitin antibody or antibody of a certain protein tag
    For example:
    Anti-Ub, raised in our laboratory
    Anti-His (Santa Cruz, catalog number: sc-0836 )
    Nickel-HRP (KPL, Kirkegaard & Perry Laboratories, catalog number: 24-01-01 )
    Anti-GST (Beijing Protein Innovation, catalog number: AbM59001-2H5-PU )
    Anti-MBP (New England Biolabs, catalog number: E8030S )
  9. Amylose resin (New England Biolabs, catalog number: E8021 )
  10. DTT
  11. SDS-PAGE gel
  12. Tris
  13. PMSF
  14. Skimmed milk powder or BSA
  15. Chemiluminescent HRP substrate kit (EMD Millipore, catalog number: WBKLS0100 )
  16. Glycerol
  17. Bromophenol blue
  18. MBP (maltose binding protein) Column buffer (see Recipes)
  19. 20x reaction buffer (see Recipes)
  20. 4x SDS sample buffer with or without DTT (or β- mercaptoethanol) (see Recipes)
  21. 1x PBS (see Recipes)

Equipment

  1. Centrifuge
  2. Thermo-mixturer (Eppendorf Thermomixer comfort)
  3. Protein electrophoresis apparatus
  4. Western blot apparatus

Procedure

  1. DTT sensitive thio-ester assay of E2 protein
    1. The reaction is performed in total 30 μl, including 1.5 μl of 20x buffer, 50 ng of E1, 200-500 ng E2, and 2 μg ubiquitin.
    2. Incubate the reactions at 37 °C for 5 min.
    3. Split the reactions by adding 10 μl 4x SDS sample buffer with or without DTT (or β- mercaptoethanol).
    4. Boil the samples at 100 °C for 5 min.
    5. The reaction products are separated with 12% SDS-PAGE gel and detected by Western blotting with anti-ub antibody or antibody for certain tag fused with the E2 protein to detect the formation of DTT-sensitive thio-ester bonds.
      1. The sample proteins separated by 12% SDS-PAGE gel are electroblotted to nitrocellulose membrane at 100 V for 75 min.
      2. The membrane is blocked with 1x PBS containing 5% skimmed milk powder for 1 h at room temperature.
      3. The membrane was then incubated first with primary antibody (suggested dilution ratio: 1:5,000 for anti-Ub, 1:500 for anti-His, 1:500 for anti-MBP antiserum) then with secondary antibody diluted in 1x PBS containing 3% skimmed milk for 1 h at room temperature. Wash the membrane with 1x PBS for two times (15 min each) after it was incubated with the primary and secondary antibody. If detect His tagged protein with Nickel-HRP,just incubate the membrane with 1:15,000 dilution of Nickel-HRP in 1x PBS containing 1% BSA for 1 h, wash the membrane two times and then bands can be detected.
      4. Bands were detected with the Millipore chemiluminescent HRP substrate kit. (Figure 1)


        Figure 1. DTT sensitive thio-ester assay of His-UBC3 ((Kraft et al., 2005)). The reaction samples were detected with anti-His antibody. The arrows indicate the DTT-sensitive thioester linkage. The open triangles indicate bands of E2 protein itself that was not attached to Ub. Asterisks indicate His-tagged Ub.

  2. Autoubiquitination assay of E3 protein
    The E3 proteins can be purified in a 1.5-ml Eppendorf tube from cell crude extract just before use (recombinant MBP (Maltose binding protein)-E3 protein is used as an example below).
    1. Vortex and thoroughly suspend the amylose beads.
    2. Aliquot 100 μl of bead suspension to a sterile microcentrifuge tube.
    3. Add 1 ml of MBP column buffer and resuspend the beads.
    4. Centrifuge at 400 x g for 2 min and decant supernatant. Repeat wash.
    5. Add 0.5-1 ml of crude extract (the total amount of the E3 protein should be 0.5-1 μg) to the tube containing the prewashed beads.
    6. Rotate at room temperature for 1 h (or 4 °C for 2 h).
    7. Wash the beads with 1 ml of 50 mM Tris–HCl (pH 7.5) for three times (like steps B3-4) and remove all the liquid of the final wash using very thin tips.
    8. Prepare the reactions in total 30 μl, including 1.5 μl of 20x reaction buffer, 50 ng of E1, 200–500 ng of E2, and 5 μg of ubiquitin. Add the reaction system to the tubes containing the amylose resin beads binding with MBP-E3 proteins.
    9. The reactions minus E1 and minus E2 respectively should be performed at the same time as control.
      1. Incubate the reactions at 30 °C for 1.5 h with agitation (900 rpm) in a thermomixer.
      2. Split the reactions by adding 10 μl 4x SDS sample buffer (with DTT or β- mercaptoethanol) and boil the samples at 100 °C for 5 min.
      3. The reaction products are separated with 8–12% SDS-PAGE gel and detected with anti-ubiquitin antibody or antibody for certain tag fused with ubiquitin or anti-MBP antibody by Western blotting. (see steps A5a-d) (Figure 2)


        Figure 2. E3 ligase autoubiquitination activity of MBP-SDIR1 (Zhang et al., 2007).
        ).
        MBP-SDIR1 was assayed for E3 activity in the presence of E1 (from wheat), E2 (UBCH5b) and 6x His tagged ubiquitin. Samples were resolved by 8% SDS-PAGE. The nickel–horseradish peroxidase (Nickel-HRP) was used to detect His tag ubiquitin.
        Note: E2/E3 specificities could be explored using different E2 proteins combined with the same E3 in this assay.

  3. E3/substrate ubiquitination assay
    The E3 and substrate protein should be fused with different tag (and the tag also should be different with the tag fused with E1, E2 and ubiquitin) and the recombinant proteins should be expressed and purified before use. The proteins also can be prepared via in vivo expression such as agroinfiltration in Nictotiana benthamiana.
    1. Prepare the reactions in total 30 μl, including 1.5 μl of 20x reaction buffer, 50 ng of E1, 200 ng of E2, 200-500 ng E3, 500 ng substrate proteins and 5 μg of ubiquitin. The reactions minus E1, minus E2 (and minus E3) respectively should be performed at the same time as control.
    2. Incubate the reactions at 30 °C for 1.5 h.
    3. Split the reactions by adding 10 μl 4x SDS sample buffer (with DTT or β- mercaptoethanol) and boil the samples at 100 °C for 5 min.
    4. The reaction products are separated with 8–12% SDS-PAGE gel and detected with anti-ubiquitin antibody or antibody for certain tag fused with substrate protein by Western blotting. (see steps A5a-d) (Figure 3)


      Figure 3. HY5-GFP (substrate protein) was ubiquitinated by Myc-COP1 (E3 ligase) (Zhao et al., 2013). HY5-GFP and Myc-COP1 were all expressed via agroinfiltration. Then, the cell lysates were mixed and immunoprecipitated with anti-Myc antibody. The immunoprecipitated product was applied for a further in vitro ubiquitination assay. E1 (from wheat), E2 (UBCH5b) and 6x His tagged Ubiquitin (Ub) were added to the reaction. ★represents the mixture of poly-ubiquitinated HY5-GFP and Myc-COP1. ▲means mono-ubiquitinated E2.
      (a) The in vitro ubiquitination samples were detected by immunoblot with anti-GFP antibody.
      (b) The in vitro ubiquitination samples were detected by immunoblot with Nickel-HRP (or anti-His antibody) to detect His-Ubiquitin.
      Note: The E3 proteins can be purified in a 1.5-ml Eppendorf tube from cell crude extract just before use as steps B1-5. And then the following E3-substrate ubiquitination reactions should be performed with agitation (900 rpm) in a thermomixer.

Recipes

  1. MBP column buffer
    20 mM Tris-HCl (pH 7.4)
    0.2 M NaCl
    1 mM EDTA
    Add 1 mM DTT and 1 mM PMSF before use
  2. 20x reaction buffer
    1 M Tris pH 7.5
    40 mM ATP
    100 mM MgCl2
    40 mM DTT
    Note: Aliquoted and stored at -20 °C. Take out an aliquot from -20 °C just before use and each aliquot can be used only once.
  3. 4x SDS sample buffer
    0.2 M Tris pH 6.8
    8% SDS
    40% glycerol
    0.004% bromophenol blue
    0.4 M DTT (or 20% β-mercaptoethanol)
    The buffer without DTT or β-mercaptoethanol can be prepared according to this recipe
  4. 1x PBS
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    2 mM KH2PO4
    Adjust to pH 7.4

Acknowledgments

This protocol was developed from the following published paper: Zhao et al. (2013). This work was supported by grants from the National Basic Research Program of China (973 Program) (2011CB915402) and the National Science Foundation of China (CNSF 31030047/90717006). Zhao QZ is supported by National Science Foundation of China grant CNSF 31200907.

References

  1. Kraft, E., Stone, S. L., Ma, L., Su, N., Gao, Y., Lau, O. S., Deng, X. W. and Callis, J. (2005). Genome analysis and functional characterization of the E2 and RING-type E3 ligase ubiquitination enzymes of Arabidopsis. Plant Physiol 139(4): 1597-1611.
  2. Liu, L., Zhang, Y., Tang, S., Zhao, Q.,Zhang, Z., Zhang, H., Li, D., Guo, H. and Xie, Q. (2010). An efficient system to detect protein ubiquitination by agroinfiltration in Nictotiana benthamiana. Plant J 61(1): 893-533.
  3. Zhang, Y., Yang, C., Li, Y., Zheng, N., Chen, H., Zhao, Q., Gao, T., Guo, H. and Xie, Q. (2007). SDIR1 is a RING finger E3 ligase that positively regulates stress-responsive abscisic acid signaling in Arabidopsis. Plant Cell 19(6): 1912-1929.
  4. Zhao, Q., Tian, M., Li, Q., Cui, F., Liu, L., Yin, B. and Xie, Q. (2013). A plant-specific in vitro ubiquitination analysis system. Plant J 74(3): 524-533. 

简介

泛素可以通过泛素激活酶(E1),泛素缀合酶(E2)和泛素蛋白连接酶(E3)的协同作用作为蛋白标签添加到底物蛋白中。 在E1和泛素的存在下,E2活性可以通过硫酯形成来确定。 推定的蛋白质的E3活性以及E2/E3或E3 /底物特异性也可以通过体外泛素化测定来研究。 结果可以通过用某些抗体的western印迹来检测。 在该测定中总是使用从细菌系统表达的纯化蛋白质。

关键字:泛素化, 体外, 泛素连接酶, 泛素结合酶

材料和试剂

  1. 细胞粗提取物
  2. 纯化蛋白或泛素活化酶粗提物(E1)
  3. 与蛋白质标签(例如6x His标签)融合的纯化的重组泛素偶联酶(E2)
  4. 纯化的重组泛素连接酶(E3)
  5. 纯化的泛素或与标签融合的重组泛素蛋白(< kraft 等人,2005; Liu等人,2010)
  6. ATP(Sigma-Aldrich,目录号:A7699)
  7. MgCl 2(Sigma-Aldrich,目录号:V900020)
  8. 抗遍在蛋白抗体或某种蛋白质标签的抗体
    例如:
    抗Ub,在我们的实验室提高
    Anti-His(Santa Cruz,目录号:sc-0836)
    Nickel-HRP(KPL,Kirkegaard& Perry Laboratories,目录号:24-01-01)
    抗GST(北京蛋白质创新,目录号:AbM59001-2H5-PU)
    抗MBP(New England Biolabs,目录号:E8030S)
  9. 直链淀粉树脂(New England Biolabs,目录号:E8021)
  10. DTT
  11. SDS-PAGE凝胶
  12. Tris
  13. PMSF
  14. 脱脂奶粉或BSA
  15. 化学发光HRP底物试剂盒(EMD Millipore,目录号:WBKLS0100)
  16. 甘油
  17. 溴酚蓝
  18. MBP(麦芽糖结合蛋白)柱缓冲液(参见配方)
  19. 20x反应缓冲液(见配方)
  20. 4x SDS样品缓冲液,含或不含DTT(或β-巯基乙醇)(参见配方)
  21. 1x PBS(请参阅配方)

设备

  1. 离心机
  2. 热混合器(Eppendorf Thermomixer comfort)
  3. 蛋白电泳仪
  4. Western印迹仪

程序

  1. E2蛋白的DTT灵敏硫酯测定
    1. 反应总共进行30μl,包括1.5μl20x缓冲液,50ng E1,200-500ng E2和2μg泛素。
    2. 孵育反应在37℃下5分钟
    3. 通过加入10μl含或不含DTT(或β-巯基乙醇)的4x SDS样品缓冲液分离反应物
    4. 将样品在100℃下煮沸5分钟
    5. 反应产物用12%SDS-PAGE凝胶分离,并通过用针对与E2蛋白融合的某些标签的抗ub抗体或抗体进行Western印迹来检测DTT敏感的硫酯键的形成。
      1. 将通过12%SDS-PAGE凝胶分离的样品蛋白质在100V下电印迹至硝酸纤维素膜75分钟。
      2. 用含有5%脱脂奶粉的1×PBS在室温下封闭膜1小时。
      3. 然后首先用第一抗体(建议的稀释比例:抗-Ub为1:5,000,抗His为1:500,抗MBP抗血清为1:500)然后用在含有3%胎牛血清的1×PBS中稀释的第二抗体脱脂牛奶在室温下1小时。在与初级和二级抗体温育后,用1x PBS洗涤膜两次(每次15分钟)。如果用镍-HRP检测His标记的蛋白,只需将膜与1:15,000稀释的镍-HRP在含有1%BSA的1×PBS中孵育1小时,洗涤膜两次,然后可以检测条带。
      4. 用Millipore化学发光HRP底物试剂盒检测条带。 (图1)


        图1.His-UBC3的DTT敏感性硫酯测定((Kraft等人,2005))。用抗His抗体检测反应样品。箭头表示DTT敏感的硫酯键。空心三角形表示E2蛋白本身的条带,其没有连接到Ub。星号表示带有His标签的Ub
  2. E3蛋白的自身泛素化测定
    E3蛋白可以在即将使用之前从细胞粗提取物的1.5-ml Eppendorf管中纯化(重组MBP(麦芽糖结合蛋白)-E3蛋白用作下面的实例)。
    1. 涡旋并彻底悬浮直链淀粉珠
    2. 等分100微升珠悬浮液到无菌微量离心管
    3. 加入1ml MBP柱缓冲液,并重悬浮珠。
    4. 在400×g离心2分钟,倾析上清液。 重复清洗。
    5. 向含有预洗珠子的管中加入0.5-1ml粗提取物(E3蛋白的总量应为0.5-1μg)。
    6. 在室温下旋转1小时(或4℃,2小时)
    7. 用1ml 50mM Tris-HCl(pH 7.5)洗涤珠子三次(如步骤B3-4),并使用非常薄的尖端除去最后一次洗涤的所有液体。
    8. 准备反应总共30微升,包括1.5微升20x反应缓冲液,50纳克E1,200-500纳克E2和5微克泛素。 将反应系统添加到含有与MBP-E3蛋白结合的直链淀粉树脂珠的管中。
    9. 反应分别减去E1和负E2应与控制同时进行。
      1. 在30℃下在热混合器中搅拌(900rpm)孵育反应1.5小时
      2. 通过加入10μl4x SDS样品缓冲液(用DTT或β-巯基乙醇)分裂反应,并将样品在100℃煮沸5分钟。
      3. 反应产物用8-12%SDS-PAGE凝胶分离,用抗泛素抗体或抗体通过Western印迹检测与泛素或抗MBP抗体融合的某些标签的抗体。 (参见步骤A5a-d)(图2)


        图2.MBP-SDIR1的E3连接酶自身遍在蛋白化活性(Zhang等人,2007)。
        )。在E1(来自小麦),E2(UBCH5b)和6x His标记的遍在蛋白的存在下,测定MBP-SDIR1的E3活性。通过8%SDS-PAGE分离样品。使用镍 - 辣根过氧化物酶(镍-HRP)检测His标签遍在蛋白。
        注意:在此测定中,可以使用与相同E3组合的不同E2蛋白来研究E2/E3特异性。
  3. E3 /底物泛素化测定
    E3和底物蛋白应与不同的标签融合(标签也应与E1,E2和泛素融合的标签不同),重组蛋白应在使用前进行表达和纯化。蛋白质也可以通过体内表达来制备,例如本氏烟草中的农杆菌浸润。
    1. 准备反应总共30微升,包括1.5微升20x反应缓冲液,50纳克E1,200纳克E2,200-500纳克E3,500纳克底物蛋白和5微克泛素。反应减去E1,减去E2(和减E3)应分别与控制同时进行
    2. 在30°C孵育反应1.5小时
    3. 通过加入10μl4x SDS样品缓冲液(用DTT或β-巯基乙醇)分裂反应,并将样品在100℃煮沸5分钟。
    4. 反应产物用8-12%SDS-PAGE凝胶分离,并用抗泛素抗体或抗体通过Western印迹检测与底物蛋白融合的某些标签。 (参见步骤A5a-d)(图3)


      图3.HY5-GFP(底物蛋白)被Myc-COP1(E3连接酶)泛素化(Zhao等人,2013)。 HY5-GFP和Myc-COP1都通过农杆菌浸润表达。然后,将细胞裂解物混合并用抗-Myc抗体免疫沉淀。将免疫沉淀产物用于进一步的体外泛素化测定。 E1(来自小麦),E2(UBCH5b)和6×His标记的泛素(Ub)。 ★代表多泛素化HY5-GFP和Myc-COP1的混合物。 ▲表示单泛素化E2。
      (a)通过用抗GFP抗体的免疫印迹检测体外泛素化样品。
      (b)通过用镍-HRP(或抗His抗体)的免疫印迹检测体外泛素化样品,以检测His-遍在蛋白。
      注意:在用作步骤B1-5之前,E3蛋白可以在来自细胞粗提物的1.5-ml Eppendorf管中纯化。然后在热混合器中用搅拌(900rpm)进行以下E3-底物泛素化反应。

食谱

  1. MBP列缓冲区
    20mM Tris-HCl(pH7.4) 0.2 M NaCl
    1mM EDTA
    在使用前加入1 mM DTT和1 mM PMSF
  2. 20x反应缓冲液
    1M Tris pH 7.5
    40 mM ATP
    100mM MgCl 2/v/v 40 mM DTT
    注意:等分并保存在-20°C。 在使用前从-20°C取出等分试样,每个等分试样只能使用一次。
  3. 4x SDS样品缓冲液
    0.2 M Tris pH 6.8
    8%SDS
    40%甘油 0.004%溴酚蓝
    0.4M DTT(或20%β-巯基乙醇) 没有DTT或β-巯基乙醇的缓冲液可以根据该配方制备
  4. 1x PBS
    137 mM NaCl 2.7 mM KCl
    10mM Na 2 HPO 4
    2mM KH 2 PO 4 sub/
    调整至pH 7.4

致谢

该协议从以下发表的文章开发: Zhao (2013)。 这项工作得到中国国家基础研究计划(973计划)(2011CB915402)和中国国家科学基金会(CNSF 31030047/90717006)的资助。 Zhao QZ获得中国国家科学基金资助中心授予CNSF 31200907。

参考文献

  1. Kraft,E.,Stone,S.L.,Ma,L.,Su,N.,Gao,Y.,Lau,O.S.,Deng,X.W.and Callis,J.(2005)。 拟南芥的E2和RING型E3连接酶泛素化酶的基因组分析和功能表征。植物生理学 139(4):1597-1611。
  2. Liu,L.,Zhang,Y.,Tang,S.,Zhao,Q.,Zhang,Z.,Zhang,H.,Li,D.,Guo,H.and Xie,Q。(2010)。 在本土烟草中通过农杆菌浸润检测蛋白质泛素化的有效系统。 Plant J 61(1):893-533。
  3. Zhang,Y.,Yang,C.,Li,Y.,Zheng,N.,Chen,H.,Zhao,Q.,Gao,T.,Guo,H.and Xie,Q。(2007)。 SDIR1是一种RING指E3连接酶,可以在拟南芥中积极调节应激反应性脱落酸信号。植物细胞 19(6):1912-1929。
  4. Zhao,Q.,Tian,M.,Li,Q.,Cui,F.,Liu,L.,Yin,B.and Xie,Q.(2013)。 植物特异性体外泛素化分析系统。 Plant J 74(3):524-533。 
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhao, Q. and Xie, Q. (2013). In vitro Protein Ubiquitination Assays. Bio-protocol 3(19): e928. DOI: 10.21769/BioProtoc.928.
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