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One-step Affinity Purification of the Chloroplast ClpP Complex from the Green Alga Chlamydomonas reinhardtii Using the Strep-tagII Epitope Tag
莱茵衣藻中的叶绿体ClpP复合体的纯化- Strep-tagII融合亲和纯化   

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Abstract

This protocol describes the affinity purification of the chloroplast ClpP complex from Chlamydomonas reinhardtii. To this purpose, we have created a Chlamydomonas reinhardtii strain in which the chloroplast encoded ClpP1 subunit of the ClpP complex is tagged at its C-terminal end by a strep-tagII peptide.

Materials and Reagents

  1. Strain: The strain ClpP1-Strep has been described in (Derrien et al., 2012) and can be obtained from the ChlamyStation culture collection (http://chlamystation.free.fr)
  2. StrepTrap HP 1 ml (GE Healthcare, catalog number: 28-9075-46 )
  3. EDTA free protease inhibitors (Roche, catalog number: 05 056 489 001 )
  4. d-Desthiobiotin ≥98% (IBA, catalog number: 2-1000-005 )
  5. 1 M Tris HCl pH 8.0 at 4 °C
  6. Ammonium sulphate saturated solution (>4.5 M; undissolved salt will settle to the bottom)
  7. 0.4 M EDTA (pH 8.0)
  8. 5 M NaCl 
  9. DTT
  10. 1 M MgCl2
  11. Glycerol
  12. Buffer A (see Recipes)
  13. Buffer B (see Recipes)
  14. Elution buffer (see Recipes)

Equipment

  1. Beckman centrifuge, rotors ( JA-10 , JA-20 , JA-12 and Type 70ti ) and polycarbonate centrifuge tubes (Beckman Coulter)
  2. Semi-continuous centrifuge (e.g. Firlabo)
  3. FPLC apparatus (BioLogic DuoFlow, Bio-Rad Laboratories)
  4. French Press (Aminco)
  5. Rotary wheel

Procedure

  1. Chlamydomonas reinhardtii cell culture
    1. Grow 5 L of a Chlamydomonas reinhardtii culture (strain) in TAP medium (Recipe available at the Chlamydomonas Resource Center http://chlamycollection.org/media-recipes/) under constant illumination (40 μE/m2/s) at 25 °C, in a 5 L stirred Erlenmeyer flasks with air bubbling. Harvest cells when culture density corresponds to late exponential phase (cell density comprised between 6 x 106 and 107 cells/ml). 5 L cultures are inoculated from pre-cultures in exponential grows phase (2 to 3 x 106 cells/ml). Cell density is measured by cell counting using a Malassez counting chamber. Knowing that Chlamydomonas generation time is ~8 h in the indicated conditions, you are able to determine the volume of the inoculum to obtain the desired concentration at the desired date and time.

  2. Harvest cells
    From here on, work at 4 °C with all solutions and glassware pre-cooled to 4 °C
    1. Concentrate cell culture by continuous centrifugation. In our Firlabo apparatus, the final volume is 1.5 L. Distribute this volume into three 500 ml centrifuge bottles.
    2. Centrifuge at 5,000 rpm for 5 min at 4 °C in a Beckman JA-10 rotor.
    3. Resuspend cells in a final volume of 500 ml of buffer A.
    4. Centrifuge at 5,000 rpm for 5 min at 4 °C in a Beckman JA-10 rotor.
    5. Resuspend cells using 10 ml of buffer A. At this stage, the solution is dense with a dark green color. Add proteases inhibitors according to the recommendations provided by Roche, and EDTA at a final concentration of 1 mM. Adjust final volume to 40 ml.

  3. Prepare soluble cellular protein extract
    1. Cell lysis: Fill up the pressure cell of the French Press with the 40 ml of cell suspension. To remove air from the pressure cell, tilt it in order to allow bubbles to reach the exit tap, open tap and push the piston until all the air has exited. Then close the tap and put the French Press under pressure (6,000 psi). Start cell lysis by slowly opening the tap of the pressure cell. Collect outflow in a 50 ml Falcon tube held on ice. During the process, liquid should flow slowly, to avoid sudden pressure drops.
    2. Repeat this procedure once on the cell lysate.
    3. Pre-clearing: transfer cell lysate into a centrifuge tube (Beckman Thickwall Polycarbonate, 50 ml, 29 x 104 mm) and centrifuge at 20,000 rpm for 30 min at 4 °C in a Beckman JA-20 rotor. Collect supernatant.
    4. Ultracentrifugation: adjust to a final volume of 50 ml with buffer A. Add MgCl2 to a final concentration of 6 mM (a minimal MgCl2 concentration of 5 mM is required to allow stacking of thylakoid membranes, needed for their complete removal). Distribute the 50 ml in two ultracentrifuge tubes (Beckman capped tube, Polycarbonate, 26.3 ml, 25 x 89 mm). Centrifuge at 60,000 rpm for 60 min at 4 °C in a Beckman rotor Type 70ti.
    5. Carefully collect supernatant (orange-yellow color) with a syringe equipped with a needle long enough to reach the bottom of the tube. Then transfer it in a 100 ml measuring cylinder.

  4. Fractionate soluble protein fraction by ammonium sulphate precipitation.
    Note: This part of the protocol is specific to Chlamydomonas reinhardtii ClpP complex, if you want to adapt the protocol to another species, you have to determine the ammonium sulphate concentration range in which your protein complex of interest precipitates.
    1. Measure the supernatant volume and add 1/3 volume of saturated ammonium sulphate solution to reach 25% saturation final. To avoid uneven precipitation, it is important to proceed progressively and under constant and gentle steering (place magnetic stir bar at bottom of cylinder). Then let the solution equilibrate for 15 min at 4 °C and transfer to a 50 ml Falcon tube.
    2. Centrifuge at 5,000 rpm for 10 min at 4 °C in a Beckman JA-12 rotor.
    3. Carefully pour off supernatant in a measuring cylinder (100 ml). Measure its volume and add 1/4 volume of saturated ammonium sulphate solution to reach 45% saturation final, taking the same precautions as above. Let the solution equilibrate for 15 min at 4 °C and transfer it into two 50 ml Falcon tube.
    4. Centrifuge at 5,000 rpm for 10 min at 4 °C in a Beckman JA-12 rotor. Carefully pour off supernatant (pellet is soft). Then using a syringe and a needle, remove all remaining traces of supernatant.
    5. Add 4 ml of buffer B onto each pellet and close caps. Put tubes on a rotary wheel at 4 °C and let the pellets solubilize (20-30 min at 20-30 rpm). Pool the two protein solutions into a 15 ml tube, if necessary adjust volume to 10 ml with buffer B and gently mix by inverting the tube.

  5. Affinity chromatography on StrepTrap column.
    This step can be performed using a FPLC apparatus, a peristaltic pump or even manually with a syringe.
    1. Equilibrate the column in buffer B (10 column volume at 1 ml/min).
    2. During this time, centrifuge the protein extract at 5,000 rpm for 5 min at 4 °C in a Beckman JA-20 rotor to remove potential insoluble particles that might clog the column.
    3. Binding: load the protein extract on the column (0.5 ml/min; if column is operated manually, this corresponds to a flow of 1 drop per second)
    4. Wash the column with buffer B (10 column volume at 1 ml/min).
    5. Elute with buffer E (10 column volume at 1ml/min, fractionation: 1ml). Collect in fractions of 1 ml. Fraction 2 corresponds to the peak of ClpP concentration, but fractions 2 to 5 can be pooled to yield high purity ClpP complex (for quality control, check Figure 1 in Derrien et al. (2012), showing samples at each step of the purification process).

  6. Concentration and storage.
    1. Once concentrated, purified ClpP complex can be stored at -80 °C for months. First, equilibrate a Centricon centrifugal filter unit (100 kDa cut-off) with 1 ml of buffer B. Then, pool elution fractions 2 to 5 and reduce volume to about 500 μl, add 500 μl of buffer B and homogenise the solution by pipeting gently (avoid bubble formation). If removal of Desthiobiotine is needed repeat this step several times in order to dilute the compound. Finally, concentrate solution to a volume of 200 μl to 300 μl. Concentration should be around 500 μg/ml. (For quality control, check Figure 3 in  Derrien et al. (2012), showing a Coomassie stained and a silver stained SDS-PAGE of the purified ClpP complex).

Recipes

  1. Buffer A
    20 mM Tris-HCl pH 8.0 at 4 °C
    150 mM NaCl
    1 mM DTT
  2. Buffer B
    20 mM Tris-HCl pH 8.0 at 4 °C
    150 mM NaCl
    10% glycerol
    1 mM DTT
  3. Elution buffer
    20 mM Tris-HCl pH 8.0 at 4 °C
    150 mM NaCl
    10% glycerol
    2.5 mM Desthiobiotine
    2.5 mM Desthiobiotine
    Note: It is important to adjust pH of the Tris HCl 1M stock solution at 4 °C. The pH of Tris buffers increases as temperature decreases (a 20 degree fall corresponds to an increase of 0.5 to 0.6 pH units)

Acknowledgments

This protocol is adapted from and previously used in Derrien et al. (2012).

References

  1. Derrien, B., Majeran, W., Effantin, G., Ebenezer, J., Friso, G., van Wijk, K. J., Steven, A. C., Maurizi, M. R. and Vallon, O. (2012). The purification of the Chlamydomonas reinhardtii chloroplast ClpP complex: additional subunits and structural features. Plant Mol Biol 80(2): 189-202.

简介

该方案描述了来自莱茵衣藻的叶绿体ClpP复合物的亲和纯化。 为此,我们已经创建了一种衣原体衣藻菌株,其中叶绿体编码的ClpP复合物的ClpP1亚基在其C末端由strep-tagII肽标记。

材料和试剂

  1. 菌株:菌株ClpP1-Strep已经在(Derrien等人,2012)中描述,并且可以从ChlamyStation培养物保藏中心获得(http://chlamystation.free.fr
  2. StrepTrap HP 1ml(GE Healthcare,目录号:28-9075-46)
  3. EDTA游离蛋白酶抑制剂(Roche,目录号:05 056 489 001)
  4. d-Desthiobiotin≥98%(IBA,目录号:2-1000-005)
  5. 1M Tris-HCl pH8.0,在4℃下
  6. 硫酸铵饱和溶液(> 4.5M;未溶解的盐将沉降到底部)
  7. 0.4 M EDTA(pH 8.0)
  8. 5 M NaCl
  9. DTT
  10. 1 M MgCl 2
  11. 甘油
  12. 缓冲液A(参见配方)
  13. 缓冲液B(参见配方)
  14. 洗脱缓冲液(见配方)

设备

  1. Beckman离心机,转子(JA-10,JA-20,JA-12和Type 70ti)和聚碳酸酯离心管(Beckman Coulter)
  2. 半连续离心机(如 Firlabo)
  3. FPLC装置(BioLogic DuoFlow,Bio-Rad Laboratories)
  4. 法语Press(Aminco)
  5. 旋转轮

程序

  1. 衣藻(Chlamydomonas reinhardtii)细胞培养物
    1. 在恒定照明(40μE/m)下在TAP培养基(可从Chlamydomonas资源中心获得的Recipe中获得5L的莱茵衣藻培养物(菌株)在25℃下,在具有空气鼓泡的5L搅拌的锥形烧瓶中。当培养物密度对应于晚指数期(细胞密度在6×10 6和10 7个细胞/ml之间)时收获细胞。从指数生长期(2至3×10 6个细胞/ml)的预培养物接种5L培养物。通过使用Malassez计数室的细胞计数来测量细胞密度。知道衣藻产生时间在指定条件下约8小时,您可以确定接种物的体积,以在所需的日期和时间获得所需的浓度。

  2. 收获单元
    从此开始,在4°C下工作,所有溶液和玻璃器皿预冷却至4°C
    1. 通过连续离心浓缩细胞培养物。在我们的Firlabo设备中,最终体积为1.5L。将该体积分配到三个500ml离心瓶中。
    2. 在4℃下在Beckman JA-10转子中以5,000rpm离心5分钟
    3. 将细胞重悬在最终体积为500ml的缓冲液A中
    4. 在4℃下在Beckman JA-10转子中以5,000rpm离心5分钟
    5. 使用10ml缓冲液A重悬细胞。在该阶段,溶液是致密的,具有深绿色。根据由Roche提供的建议加入蛋白酶抑制剂,和EDTA,终浓度为1mM。将最终体积调整为40 ml
  3. 制备可溶性细胞蛋白提取物
    1. 细胞裂解:用40ml细胞悬浮液填充French Press的压力室。为了从压力室中排出空气,倾斜它以便允许气泡到达出口,打开龙头并推动活塞,直到所有的空气都已经排出。然后关闭水龙头,将压榨机置于压力(6,000 psi)下。通过缓慢打开压力盒的水龙头开始细胞裂解。收集流出物在冰上保持的50ml Falcon管。在此过程中,液体应缓慢流动,以避免突然的压力下降
    2. 对细胞裂解液重复该程序一次。
    3. 预清洗:将细胞裂解液转移到离心管(Beckman Thickwall Polycarbonate,50ml,29×104mm)中,并在Beckman JA-20转子中在4℃下以20,000rpm离心30分钟。收集上清液。
    4. 超速离心:用缓冲液A调节至终体积为50ml。加入MgCl 2至终浓度为6mM(需要5mM的最小MgCl 2浓度为5mM)允许堆积类囊体膜,需要它们完全去除)。在两个超速离心管(Beckman加盖管,聚碳酸酯,26.3ml,25×89mm)中分配50ml。在Beckman转子70ti型中,在4℃下以60,000rpm离心60分钟
    5. 用装有足够长的针头的注射器小心地收集上清液(橙黄色)以达到管的底部。然后将其转移到100毫升的量筒中
  4. 通过硫酸铵沉淀分级分离可溶性蛋白级分 注意:这部分协议是专门针对莱茵衣藻ClpP复杂,如果你想适应协议到另一个物种,你必须确定硫酸铵浓度范围,你感兴趣的蛋白质复合物沉淀。 >
    1. 测量上清液体积,加入1/3体积的饱和硫酸铵溶液,最终达到25%饱和度。为了避免不均匀沉淀,重要的是逐渐进行并在恒定和温和的转向(将磁力搅拌棒放置在圆筒底部)。然后让溶液在4℃平衡15分钟,并转移到50ml Falcon管。
    2. 在4℃下在Beckman JA-12转子中以5,000rpm离心10分钟
    3. 小心地将上清液倒入量筒(100ml)中。测量其体积,加入1/4体积的饱和硫酸铵溶液以达到最终的45%饱和度,采取与上述相同的预防措施。让溶液在4℃平衡15分钟,并将其转移到两个50ml Falcon管。
    4. 在4℃下在Beckman JA-12转子中以5,000rpm离心10分钟。小心地倒出上清液(沉淀物是软的)。然后使用注射器和针头,清除所有剩余的上清液。
    5. 加入4毫升缓冲液B的每个丸和封盖。 将管置于4℃的旋转轮上,使丸粒溶解(20-30rpm下20-30分钟)。 将两种蛋白质溶液混合到15ml试管中,如果需要,用缓冲液B调节体积至10ml,通过颠倒管子轻轻混合。

  5. 在StrepTrap柱上进行亲和层析 该步骤可以使用FPLC装置,蠕动泵或甚至用注射器手动进行。
    1. 在缓冲液B(10柱体积,1ml/min)中平衡该柱
    2. 在此期间,在4℃下在Beckman JA-20转子中以5,000rpm离心蛋白质提取物5分钟,以除去可能阻塞柱的可能不溶性颗粒。
    3. 结合:将蛋白质提取物装载在柱上(0.5ml/min;如果柱手动操作,这对应于每秒1滴的流动)
    4. 用缓冲液B(10柱体积,1ml/min)洗柱
    5. 用缓冲液E洗脱(10柱体积,1ml/min,分级:1ml)。以1ml的级分收集。部分2对应于ClpP浓度的峰,但可以合并部分2至5以产生高纯度ClpP复合物(用于质量控制,参见Derrien等人(2012)中的图1,显示样品在纯化过程的每个步骤)。

  6. 浓缩和储存。
    1. 一旦浓缩,纯化的ClpP复合物可以在-80℃下储存数月。首先,用1ml缓冲液B平衡Centricon离心过滤单元(100kDa截留)。然后,将洗脱级分2至5洗脱并将体积减少至约500μl,加入500μl缓冲液B,并通过吸移将溶液均化轻轻地(避免气泡形成)。如果需要除去Desthiobiotine,重复该步骤几次以稀释化合物。最后,浓缩溶液至200μl至300μl的体积。浓度应约为500μg/ml。 (对于质量控制,检查Derrien等人(2012)中的图3,显示考马斯染色和纯化的ClpP复合物的银染SDS-PAGE)。

食谱

  1. 缓冲区A
    20mM Tris-HCl pH8.0,4℃ 150mM NaCl 1 mM DTT
  2. 缓冲区B
    20mM Tris-HCl pH8.0,4℃ 150mM NaCl 10%甘油 1 mM DTT
  3. 洗脱缓冲液
    20mM Tris-HCl pH8.0,4℃ 150mM NaCl 10%甘油 2.5 mM Desthiobiotine
    2.5 mM Desthiobiotine
    < i>注意:重要的是将Tris HCl 1M储液的pH调节到4℃。 Tris缓冲液的pH随着温度降低而增加(20度下降对应于0.5至0.6个pH单位的增加)

致谢

该协议改编自并且以前在Derrien等人(2012)中使用。

参考文献

  1. Derrien,B.,Majeran,W.,Effantin,G.,Ebenezer,J.,Friso,G.,van Wijk,K.J.,Steven,A.C.,Maurizi,M.R.and Vallon, 净化莱茵衣藻叶绿体ClpP复合物:额外的亚基和结构 功能。 Plant Mol Biol 80(2):189-202
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Derrien, B. and Vallon, O. (2013). One-step Affinity Purification of the Chloroplast ClpP Complex from the Green Alga Chlamydomonas reinhardtii Using the Strep-tagII Epitope Tag. Bio-protocol 3(1): e315. DOI: 10.21769/BioProtoc.315.
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