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Expression, Purification and in vitro Enzyme Activity Assay of Plant Derived GTPase
植物源GTP酶的表达、纯化和体外酶活性的测定   

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Abstract

Based on gene expression data after biotic stress, the GTPase RabA4c has been suggested to regulate pathogen-induced callose biosynthesis in the model organism Arabidopsis thaliana. We studied the function of RabA4c in its native and dominant negative (dn) isoform. In planta, RabA4c overexpression prevented penetration of the virulent powdery mildew Golovinomyces cichoracearum into epidermal leaf cells. This penetration resistance was caused by enhanced callose deposition at sites of attempted fungal penetration at early time points of infection. By contrast, RabA4c (dn) overexpression did not increase callose deposition or penetration resistance.
In this protocol, we describe the expression, purification and activity assay of the heterologously expressed GTPase RabA4c from A. thaliana based on the publication Ellinger et al. (2014). We fused RabA4c to the fluorophore mCitrine and expressed this protein in the yeast strain Pichia pastoris GS115. For purification of RabA4c, we used the GFP-Trap_A kit (Chromo Tek) which specifically binds to GFP derivatives like mCitrine. The enzyme activity assay was done by using the GTPase Assay Kit from Innova Biosciences. In general, we followed the instructions made by the manufacturers.

Materials and Reagents

  1. Protein expression
    1. pGAPZ A, B, & C Pichia pastoris Expression Vectors (Life Technologies, catalog number: V200-20 )
      Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: V200-20”.
    2. ZeocinTM (InvivoGen, catalog number: ant-zn-5b ) for selection
    3. 1 % yeast extract (Carl Roth GmbH + Co., catalog number: 2363.3 )
    4. 2 % peptone (BD, catalog number: 211677 )
    5. D(+)-Glucose (Carl Roth GmbH + Co., catalog number: X997.3 )
    6. Yeast Peptone Dextrose (YPD) media (see Recipes)

  2. Protein purification
    1. Acid washed glass beads (Sigma-Aldrich, catalog number: G 8772 )
    2. GFP-Trap® (ChromoTek GmbH, catalog number: gta-20 )
    3. NanoOrange Protein Quantification Kit (Invitrogen, catalog number: N-6666 )
      Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: N-6666”.
    4. 10 mM Tris (pH 7.5)
    5. 50 mM NaCl
    6. 0.5 mM EDTA (pH 8.0)
    7. 100 mM Glycine (pH 2.5)
    8. 1 M Tris (pH 10.4)
    9. Dilution buffer (see Recipes)
    10. Wash buffer I (see Recipes)
    11. Wash buffer II (see Recipes)
    12. Elution buffer (see Recipes)

  3. GTPase activity assay
    1. GTPase Assay Kit (Innova Biosciences Ltd., catalog number: 602-0121 )
    2. Negative control: expressed and purified GFP using P. pastoris as expression system (plasmid pGAPZ::eGFP)

Equipment

  1. 300 ml flasks
  2. Shaker (160 rpm)
  3. Centrifuge for 50 ml Falcon tubes (3,220 x g)
  4. Vortex
  5. Tube rotator at 4 °C and room temperature
  6. Plate Reader (BioTek Instruments, model: Synergy HTX Multi-Mode Reader)
  7. Tabletop centrifuge (cooling)

Procedure

  1. Protein expression
    1. Strain: Pichia pastoris GS115
    2. Expression vector: pGAPZ
    3. Pre-culture: incubate one colony from a YPD (+ zeocin) plate in 4 ml liquid YPD (+ zeocin) media at 28 °C and 160 rpm for one day.
    4. Main-culture: Incubated 1 ml of the pre-culture in 100 ml fresh YPD (+ zeocin) media at 28 °C and 160 rpm for 3 days.
    5. Strains: pGAPZ::RabA4c-mCit[NA]/ [DN], control pGAPZ:e:GFP [NA= native form, DN= dominant negative, please refer to Ellinger et al. (2014) for strain generation].
    6. Yeast cells were transferred in 50 ml Falcon tubes and harvested by centrifugation (3,220 x g, 10 min, 4 °C).
    7. Discard the supernatant and wash the cell pellet with sterile ddH2O.
    8. Mechanical lysis: Add an identical volume of sterile acid washed glass beads to the cell pellet and vortex the beads-cell suspension for 30 sec at maximum speed followed by chilling on ice for 30 sec, repeat these steps A8-10 times.
    9. Spin down the glass beads at 453 x g at 4 °C for 2 min.
    10. Transfer the supernatant in a new 2 ml tube, wash the glass beads with 1 ml dilution buffer (see above), centrifuge again and transfer the supernatant also to the 2 ml tube.
    11. Separate the cell debris by centrifugation at max speed at 4 °C for 30 min.
    12. Transfer the clear supernatant in a new tube and store at 4 °C until starting with protein purification. Store the pellet at -20 °C for SDS-PAGE analysis.

  2. Protein purification
    1. Equilibrate 25 µl (for one sample) GFP-Trap_A agarose beads in dilution buffer (use a 2 ml tube): Wash 3 times with 500 µl ice cold dilution buffer, between the wash steps spin beads down at 719 x g at 4 °C for 2 min and discard the supernatant.
    2. Add the crude extract to the equilibrate beads and incubate the sample for 4 h at 4 °C under constant mixing (e.g., tube rotator).
    3. Spin down sample at 719 x g at 4 °C for 2 min and carefully remove the supernatant by pipetting. Store an aliquot for “non-bound proteins” analysis at 4 °C.
    4. Wash beads 3 times with 1 ml ice cold wash buffer I and II through centrifugation at 719 x g at 4 °C for 2 min; discard the supernatant.
    5. Beads fused to the protein of interest are ready-to-use for enzyme activity assay.

  3. GTPase activity assay (for more information, please refer to the GTPase Assay Kit Manual)
    GTPases [also known as G proteins (guanine nucleotide-binding proteins)] function as molecular switches that cycle between an active and inactive state. The cycle is linked to the binding and hydrolysis of GTP. In the active state, these enzymes interact with GTP to perform diverse cellular functions. Inactivation occurs through the association of GAP (GTPase-activating protein). This protein hydrolyzes GTP to GDP and free phosphate. To return to the active state, the enzyme GEF (guanine nucleotide exchange factor) is required to catalyze the exchange of GDP for GTP (Figure 1).


    Figure 1. Principle of the GTPase assay kit (Innova Bioscience)

    1. Prepare the substrate buffer mix (SB-Mix) and samples for a standard curve as described in the GTPase Assay kit Manual.
    2. Add 350 µl SB-Mix buffer to the purified beads-protein solution.
    3. Incubate samples at room temperature for 30 min under constant mixing.
    4. Spin down beads at 1,073 x g for 5 min using a tabletop centrifuge.
    5. Put 100 µl of reaction buffer in one well of a 96-well plate (triplicates) and add 100 µl pure H2O; prepare samples for the standard curve in the same way.
    6. Prepare the Gold-Mix buffer (see GTPase Assay kit Manual) and add 50 µl to each well to stop the reaction.
    7. After 2 min add 20 µl stabilizer.
    8. Incubate plate at room temperature for 30 min in the dark.
    9. Read plate at a wavelength of 650 nm (590- 660 nm, see GTPase Assay kit Manual).
    10. For calculation of enzyme activity, follow Appendix 1 of the GTPase Assay kit Manual (Figure 2).


      Figure 2. Specific enzyme activity of RabA4c-mCit [NA]/ [DN] and GFP as control. The GTPase assay was performed as described above (Procedure). Error bars represent standard deviations.

  4. SDS-PAGE and protein quantification
    1. After GTPase Assay: Elute the target protein from the GFP-Trap_A beads by adding 200 µl 100 mM glycine (pH 2.5), mix shortly and incubate at room temperature for 2 min.
    2. Add 20 µl 1 M TRIS base (pH 10.4) for neutralization.
    3. Spin beads down at 1,073 x g at room temperature for 2 min, transfer supernatant to a new tube.
    4. Protein quantification follows the instructions of the manufactures (NanoOrange Protein Quantification Kit, see above), the protein amount is necessary to determine the enzyme activity.

Recipes

  1. Yeast Peptone Dextrose (YPD) media
    1% yeast extract
    2% peptone
    D(+)-Glucose
  2. Dilution buffer
    10 mM Tris (pH 7.5)
    150 mM NaCl
    0.5 mM EDTA (pH 8.0)
  3. Wash buffer I
    10 mM Tris (pH 7.5)
    300 mM NaCl
    0.5 mM EDTA (pH 8.0)
  4. Wash buffer II
    10 mM Tris (pH 7.5)
    500 mM NaCl
    0.5 mM EDTA (pH 8.0)
  5. Elution buffer
    100 mM glycine (pH 2.5)
    1 M Tris (pH 10.4)

Acknowledgments

This work was performed as a part of the publication of Ellinger et al. (2014) and was funded by the Federal Ministry of Education and Research (BMBF) within the research focus BioEnergy 2021: "CallBio - Resistant Plants for simplified Bio-ethanol Production through optimized Biosynthesis of the Cell Wall Polymer Callose".

References

  1. Ellinger, D., Glöckner, A., Koch, J., Naumann, M., Stürtz, V., Schütt, K., Manisseri, C., Somerville, S. C. and Voigt, C. A. (2014). Interaction of the Arabidopsis GTPase RabA4c with its effector PMR4 results in complete penetration resistance to powdery mildew. Plant Cell 26(7): 3185-3200.

简介

基于生物胁迫后的基因表达数据,已经建议GTP酶RabA4c 在模拟生物拟南芥中调节病原体诱导的胼lose质生物合成。我们研究了其天然和显性负性(dn)同种型中的RabA4c 的功能。 ,RabA4c 过表达阻止了毒性白粉病菌向鸡冠状叶细胞的侵入。这种穿透阻力是由于在感染早期试图真菌穿透的部位处的胼cal质沉积增加引起的。相反,RabA4c(dn)过表达不增加胼cal质沉积或穿透抗性。在本方案中,我们描述了异源表达的GTPase RabA4c的表达,纯化和活性测定从 thaliana ,基于出版物Ellinger 等人(2014)。我们将< em> RabA4c</em>与荧光团mCitrine融合,并在酵母菌株巴斯德毕赤酵母GS115中表达该蛋白质。为了纯化RabA4c,我们使用特异性结合GFP衍生物如mCitrine的GFP-Trap_A 试剂盒(Chromo Tek)。通过使用来自Innova Biosciences的 GTPase测定试剂盒进行酶活性测定。一般来说,我们遵循制造商的指示。

材料和试剂

  1. 蛋白质表达
    1. pGAPZ A,B,毕赤酵母表达载体(Life Technologies,目录号:V200-20)
      注意:目前,它是"Thermo Fisher Scientific,Invitrogen TM ,目录号:V200-20"。
    2. 用于选择的Zeocin TM (InvivoGen,目录号:ant-zn-5b)
    3. 1%酵母提取物(Carl Roth GmbH + Co.,目录号:2363.3)
    4. 2%蛋白胨(BD,目录号:211677)
    5. D(+) - 葡萄糖(Carl Roth GmbH + Co.,目录号:X997.3)
    6. 酵母蛋白胨葡萄糖(YPD)培养基(见Recipes)

  2. 蛋白质纯化
    1. 酸洗玻璃珠(Sigma-Aldrich,目录号:G 8772)
    2. GFP-Trap (ChromoTek GmbH,目录号:gta-20)
    3. NanoOrange Protein Quantification Kit(Invitrogen,目录号:N-6666) 注意:目前,"Thermo Fisher Scientific,Molecular Probes TM ,目录号:N-6666"。
    4. 10mM Tris(pH7.5)
    5. 50mM NaCl
    6. 0.5mM EDTA(pH8.0)
    7. 100mM甘氨酸(pH 2.5)
    8. 1 M Tris(pH 10.4)
    9. 稀释缓冲液(参见配方)
    10. 洗涤缓冲液I(见配方)
    11. 洗涤缓冲液II(参见配方)
    12. 洗脱缓冲液(见配方)

  3. GTP酶活性测定
    1. GTPase测定试剂盒(Innova Biosciences Ltd.,目录号:602-0121)
    2. 阴性对照:使用p表达和纯化GFP。 pastoris作为表达系统(质粒pGAPZ :: eGFP)

设备

  1. 300 ml烧瓶
  2. 摇床(160rpm)
  3. 离心机用于50ml Falcon管(3,220×g/g)
  4. 涡流
  5. 管式旋转器在4℃和室温下
  6. Plate Reader(BioTek Instruments,型号:Synergy HTX多模式读取器)
  7. 台式离心机(冷却)

程序

  1. 蛋白质表达
    1. 菌株:

      巴斯德毕赤酵母GS115

    2. 表达载体:pGAPZ
    3. 预培养:将来自YPD(+ zeocin)平板的一个集落孵育在4ml中 液体YPD(+ zeocin)介质在28℃和160rpm下培养1天
    4. 主培养:在28℃和160rpm下,在100ml新鲜YPD(+ zeocin)培养基中培养1ml预培养物3天。
    5. 菌株:pGAPZ :: RabA4c-mCit [NA]/[DN],对照 pGAPZ:e:GFP 原生形式,DN =显性阴性,请参考Ellinger等人的(2014)应变生成]。
    6. 将酵母细胞转移到50ml Falcon管中,并通过离心(3220×g,10分钟,4℃)收获。
    7. 弃去上清液并用无菌ddH 2 O洗涤细胞沉淀
    8. 机械裂解:加入相同体积的无菌酸洗 玻璃珠到细胞沉淀并涡旋珠细胞悬浮液 以最大速度30秒,然后在冰上冷却30秒,重复 这些步骤A8-10次。
    9. 在4℃下将玻璃珠以453×g /分钟旋转2分钟
    10. 转移上清液在新的2毫升管,洗玻璃珠 用1ml稀释缓冲液(见上文),再次离心并转移 ?上清液也到2ml管
    11. 通过在4℃的最大速度离心30分钟分离细胞碎片。
    12. 转移清澈的上清液在一个新的管,并存储在4°C 直到蛋白质纯化开始。将沉淀储存在-20°C下 ?SDS-PAGE分析。

  2. 蛋白质纯化
    1. 平衡25μl(一个样品)GFP-Trap_A琼脂糖珠稀释 缓冲液(使用2ml试管):用500μl冰冷稀释液洗涤3次 缓冲液,在洗涤步骤之间,在4℃下将珠子以719×g离心2分钟 min并弃去上清液
    2. 将粗提取物加入 平衡珠子,并在4℃下在恒定下孵育样品4小时 ?混合(例如管旋转器)。
    3. 在4℃下以719×g离心样品 2分钟,通过吸移小心地除去上清液。存储 等分试样在4℃下进行"非结合蛋白"分析
    4. 洗珠3 次,用1ml冰冷的洗涤缓冲液I和II通过离心分离 719×g 在4℃下2分钟;丢弃上清液。
    5. 与感兴趣的蛋白质融合的珠子可以立即用于酶活性测定。

  3. GTP酶活性测定(更多信息,请参考GTPase测定试剂盒手册)
    GTP酶[也称为G蛋白(鸟嘌呤核苷酸结合蛋白)]用作在活性和非活性状态之间循环的分子开关。该循环与GTP的结合和水解有关。在活性状态下,这些酶与GTP相互作用以执行多种细胞功能。通过GAP(GTP酶激活蛋白)的结合发生失活。这种蛋白质将GTP水解成GDP和游离磷酸盐。为了恢复活性状态,需要酶GEF(鸟嘌呤核苷酸交换因子)来催化GTP的GDP的交换(图1)。


    图1. GTP酶测定试剂盒(Innova Bioscience)的原理

    1. 按照GTPase Assay试剂盒手册中所述制备底物缓冲液混合物(SB-Mix)和标准曲线的样品。
    2. 向纯化的珠 - 蛋白溶液中加入350μlSB-Mix缓冲液
    3. 在恒定混合下在室温下孵育样品30分钟
    4. 使用台式离心机将粉碎的珠子以1,073×g 离心5分钟
    5. 将100μl反应缓冲液置于96孔板的一个孔中 (一式三份)并加入100μl纯H 2 O;准备样品的标准 曲线以相同的方式。
    6. 准备Gold-Mix缓冲液(参见GTPase Assay试剂盒手册),并向每个孔中加入50μl以终止反应。
    7. 2分钟后加入20μl稳定剂。
    8. 孵育板在室温下在黑暗中30分钟。
    9. 读取波长为650 nm的光板(590-660 nm,参见GTPase Assay试剂盒手册)。
    10. 对于酶活性的计算,按照GTPase Assay试剂盒手册的附录1(图2)

      图2.RabA4c-mCit [NA]/[DN]和GFP作为对照的特异性酶活性。 如上所述进行GTP酶测定(程序)。误差线表示标准偏差。

  4. SDS-PAGE和蛋白质定量
    1. GTP酶测定后:从GFP-Trap_A 珠中洗脱目标蛋白 通过加入200μl100mM甘氨酸(pH 2.5),短暂混合并在37℃温育 室温下2分钟。
    2. 加入20μl1 M TRIS碱(pH 10.4)中和
    3. 在室温下将珠子以1.073×g离心2分钟,将上清液转移到新管中。
    4. 蛋白质定量按照制造商的说明书 (NanoOrange Protein Quantification Kit,见上),蛋白量 是确定酶活性所必需的。

食谱

  1. 酵母蛋白胨葡萄糖(YPD)培养基
    1%酵母提取物
    2%蛋白胨
    D(+) - 葡萄糖
  2. 稀释缓冲液
    10mM Tris(pH7.5) 150mM NaCl 0.5mM EDTA(pH8.0)
  3. 洗涤缓冲液I
    10mM Tris(pH7.5) 300 mM NaCl
    0.5mM EDTA(pH8.0)
  4. 洗涤缓冲液II
    10mM Tris(pH7.5) 500 mM NaCl
    0.5mM EDTA(pH8.0)
  5. 洗脱缓冲液
    100mM甘氨酸(pH 2.5)
    1 M Tris(pH 10.4)

致谢

这项工作作为Ellinger等人的出版物的一部分进行。 (2014),由联邦教育和研究部(BMBF)资助,研究重点是BioEnergy 2021:"CallBio-Resistant Plants for simplified bio-ethanol production through optimized Biosynthesis of Cell Wall Polymer Callose"。

参考文献

  1. Ellinger,D.,Gl?ckner,A.,Koch,J.,Naumann,M.,Stürtz,V.,Schütt,K.,Manisseri,C.,Somerville,S.C.and Voigt, 拟南芥 GTPase RabA4c 与其效应子PMR4导致对白粉病的完全穿透抗性。植物细胞26(7):3185-3200。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Glöckner, A. and Voigt, C. A. (2015). Expression, Purification and in vitro Enzyme Activity Assay of Plant Derived GTPase. Bio-protocol 5(22): e1651. DOI: 10.21769/BioProtoc.1651.
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