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Expression and Purification of the Thermus thermophilus Argonaute Protein
嗜热细菌Argonaute蛋白的表达和纯化   

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Abstract

The Argonaute protein of Thermus thermophilus (TtAgo) has recently been studied in detail. For its in vitro characterization, TtAgo was purified after heterologous expression in Escherichia coli (E. coli). As TtAgo expression is toxic, a tightly controlled system was used for protein expression. The expression strain E. coli KRX carries a chromosomal T7 RNA polymerase gene under control of a rhamnose promoter. The ago gene is expressed via an IPTG-inducible T7 promoter. This allows for tightly (double) controlled expression of (toxic) TtAgo. Here, we describe the steps required for controlled expression and purification of this toxic protein.

Materials and Reagents

  1. Expression of TtAgo in E. coli KRX
    1. Glycerol stock of Escherichia coli KRX (Promega corporation, catalog number: L3002 ) [encodes a T7 RNA polymerase gene under control of a rhamnose promoter], transformed with plasmids pRARE (EMD Millipore, plasmid of RosettaTM (DE3) Competent Cells, catalog number: 70954-3 ) [encodes tRNAs for rare codons to enhance protein translation efficiency] and pWUR702 [pCDF-1b derivative, with T. thermophilus HB27 TT_P0026 ago gene insert fused to an N-terminal strep(II)-tag, expression under control of an IPTG-inducible T7 promoter] (Addgene, catalog number: 53079 ).
      Note: The plasmid pWUR703 (Addgene, catalog number: 53082 ) can be used for expression of TtAgoDM (Double Mutant, D478A, D546A). Expression and purification of this protein is identical to TtAgo expression and purification.
    2. 1,000x chloramphenicol solution (34 mg/ml) dissolved in 100% ethanol
    3. 1,000x streptomycin solution (50 mg/ml) dissolved in MilliQ H2O
    4. 20% D-glucose solution dissolved in MilliQ H2O (sterile filtered)
    5. 20% L-rhamnose solution dissolved in MilliQ H2O (sterile filtered)
    6. 1 M IPTG dissolved in MilliQ H2O (sterile filtered)
    7. LB medium (see Recipes)

  2. Purification of TtAgo
    1. Cell pellet from o/n TtAgo expression (see above)
    2. Strep-Tactin Sepharose® 50% suspension (IBA, catalog number: 2-1201-XXX )
    3. 1.2 ml Bio-Spin® Chromatography Columns (Bio-Rad Laboratories, catalog number: 732-6008 )
    4. Buffer I (see Recipes)
    5. Buffer II (see Recipes)
    6. Buffer III (see Recipes)

Equipment

  1. Expression of TtAgo in E. coli KRX
    1. 50 ml Greiner tube
    2. 2.5 L Erlenmeyer flask
    3. Centrifuge
    4. 37 °C shaker incubator
    5. 20 °C shaker incubator
    6. Ice-water bath (water and ice mixed)

  2. Purification of TtAgo
    1. French-Press, sonicator, or equivalent for cell disruption
    2. Centrifuge

Procedure

  1. Expression of TtAgo in E. coli KRX
    1. Take 10 ml LB medium in a 50 ml Greiner tube.
    2. Add 10 μl 1,000x chloramphenicol solution.
    3. Add 10 μl 1,000x streptomycin solution.
    4. Add 200 μl 20% D-glucose solution.
    5. Inoculate the culture from the glycerol stock using a sterile pipette tip.
    6.  Incubate the culture o/n at 37 °C in a shaker incubator at 180 rpm.
    7. After o/n incubation, centrifuge culture for 10 min at 4,000 x g at room temperature.
    8. Remove the supernatant to remove any traces of D-glucose - this is important as D-glucose traces can prevent L-rhamnose uptake (and thereby represses TtAgo expression).
    9. Resuspend the cell pellet in 1 ml LB medium.
    10. Take a 2.5 L Erlenmeyer containing 1 L LB medium.
    11. Add 1 ml 1,000x chloramphenicol solution.
    12. Add 1 ml 1,000x streptomycin solution.
    13. Add resuspended cells.
    14. Incubate the culture at 37 °C in shaker incubator at 140 rpm.
    15. Monitor the OD600nm of the culture. If an OD600nm of 0.7-0.8 is reached (this takes ~3-4 h), transfer the culture to an ice-water bath to cold-shock it for 15 min. This step slows down the metabolism of E. coli and triggers expression of cold-shock proteins which may aid TtAgo folding during expression.
    16. Add 1 ml 1 M IPTG and 5 ml 20% L-rhamnose solution to the culture to induce expression.
    17. Transfer the culture to a 20 °C shaker incubator at 140 rpm for o/n (+/-16h) expression.
    18. Harvest the cells by centrifuging culture for 15 min at 6,000 x g at 4 °C.
    19. Remove the supernatant. You should typically end up with 4-5 grams of cell pellet.

  2. Purification of TtAgo
    1. If using a French Press for cell lysis: Resuspend cell pellet in Buffer I (5 ml Buffer I per gram of cell pellet) and pass cell suspension through French press two times at 16,000 psi (see Note c).
      If using a sonicator for cell lysis: Resuspend cell pellet in Buffer I (3 ml Buffer I per gram of cell pellet), and lyse the cells by sonication. Use a tip and sonication protocol suitable for lysis of large volume cell suspensions.
    2. Centrifuge for 30 min at 30,000 x g at 4 °C.
    3. Transfer the supernatant (cell free extract) to a clean tube.
    4. Prepare a Strep-Tactin Sepharose column by loading 500 μl of the 50% suspension in a 1.2 ml Bio-Spin chromatography column (final column volume is 250 μl).
    5. Wash column by loading the column three times with 500 μl Buffer I.
    6. Load the cell free extract on the column.
    7. Wash the column by loading it three times with 750 μl Buffer I.
    8. Wash the column by loading it three times with 750 μl Buffer II.
    9. Elute the protein by loading the column three times with 250 μl Buffer III, collect the flow through. Typical yields of TtAgo elution fractions are in the 0-2.5 μM range for fraction 1 and 3 and in the 5-10 μM range for fraction 2. Typical yields of TtAgoDM are in the 0-2.5 μM range for fraction 1 and 3 and in the 2.5-7.5 μM range for fraction 2.
      Notes:
      1. Depending on experiments, MgCl2 and MnCl2 concentrations can be lowered to 0.5 mM, or left out. NaCl concentration can be lowered to 0.5 M, however, TtAgo (especially at higher protein concentrations) is more stable at 1 M NaCl. At lower concentrations salt (especially bellow 250 mM), TtAgo is instable at protein concentrations higher than 5 μM).
      2. The protocol above describes purification of guide-free TtAgo. If guide co-purification is desired, replace all Buffer I in this experiment with Buffer II.
      3. Using a French Pressure cell or sonicator gives more or less the same yield – TtAgo is very stable and little to no protein will be lost during sonication. Keep in mind however, that sonication usually is less suitable for large volumes, and a protocol suitable for lysis of a large volume of cell suspension should be applied.

Representative data



Figure 1. Coomassie Brilliant Blue stained 12% SDS-PAGE gel of TtAgo (left) and TtAgoDM (right) purification samples. M: Bio-Rad precision plus protein marker. Marker band sizes are indicated in kDa. FT: Column flow through fraction of loaded cell free extract. W1: Column wash fraction with Buffer I. W2: Column wash fraction with Buffer II. EL1-EL3: Elution fractions 1-3. The protein appears as a band with a size of +/-75kDa.

Recipes

  1. LB medium (1 L)
    10 g tryptone
    5 g yeast extract
    10 g NaCl
    Fill up to 1 L with demiwater
    Set pH to 7.5 with NaOH
  2. Buffer I
    20 mM Tris-HCl (pH 8)
    1 M NaCl
    2 mM MgCl2
  3. Buffer II
    20 mM Tris-HCl (pH 8)
    1 M NaCl
    2 mM MnCl2
  4. Buffer III
    20 mM Tris-HCl (pH 8)
    1 M NaCl
    2 mM MnCl2
    2.5 mM Biotin
    Note: Biotin can be replaced with d-Desthiobiotin, which will allow for column regeneration.

Acknowledgments

This work was financially supported by a grant from the Netherlands Organization of Scientific Research (NWO) to J.v.d.O. (NWO-TOP, 854.10.003).

References

  1. Swarts, D. C., Jore, M. M., Westra, E. R., Zhu, Y., Janssen, J. H., Snijders, A. P., Wang, Y., Patel, D. J., Berenguer, J., Brouns, S. J. and van der Oost, J. (2014). DNA-guided DNA interference by a prokaryotic Argonaute. Nature 507(7491): 258-261.

简介

最近已经详细研究了嗜热栖热菌(Thermus thermophilus)( Tt Ago)的Argonaute蛋白。 为了其体外表征,在大肠杆菌中大肠杆菌( )中异源表达后纯化Δtt。 作为Tt Ago表达是有毒的,严格控制的系统用于蛋白质表达。 表达菌株em。 大肠杆菌KRX携带在鼠李糖启动子控制下的染色体T7RNA聚合酶基因。 基因通过IPTG诱导型T7启动子表达。 这允许(有毒的)Tt Ago的紧密(双)受控表达。 在这里,我们描述了控制表达和纯化这种毒性蛋白所需的步骤。

材料和试剂

  1. 在 E中表示 Tt 大肠杆菌KRX
    1. 大肠杆菌KRX(Promega公司,目录)的甘油原液 number:L3002 )[编码在a。控制下的T7RNA聚合酶基因 鼠李糖启动子],用质粒pRARE(EMD Millipore, Rosetta (DE3)感受态细胞,目录号:70954-3 [编码用于稀有密码子的tRNA以增强蛋白质翻译 效率]和pWUR702 [pCDF-1b衍生物,具有T。 嗜热链球菌 HB27 TT_P0026 基因插入片段融合至N端链霉亲和素(II) 在IPTG诱导型T7启动子控制下的表达](Addgene, 目录号:53079 )。
      注意:质粒pWUR703(Addgene,目录 编号:53082)可用于表达TtAgoDM(双突变体, D478A,D546A) 这种蛋白质的表达和纯化是相同的 到TtAgo的表达和纯化。
    2. 1000x氯霉素溶液(34mg/ml)溶于100%乙醇中
    3. 溶解于MilliQ H 2 O中的1,000x链霉素溶液(50mg/ml)
    4. 20%D-葡萄糖溶液溶解在MilliQ H 2 O(无菌过滤)中
    5. 溶解在MilliQ H 2 O中的20%L-鼠李糖溶液(无菌过滤)
    6. 溶解在MilliQ H 2 O(无菌过滤)中的1M IPTG
    7. LB培养基(参见配方)

  2. 净化 Tt Ago
    1. 来自o/n tAgo表达式的细胞沉淀(见上文)
    2. Strep-Tactin Sepharose 50%悬浮液(IBA,目录号:2-1201-XXX)
    3. 1.2ml Bio-Spin色谱柱(Bio-Rad Laboratories,目录号:732-6008)
    4. 缓冲区I(参见配方)
    5. 缓冲液II(参见配方)
    6. 缓冲液III(参见配方)

设备

  1. 在 E中表示 Tt 大肠杆菌KRX
    1. 50ml Greiner管
    2. 2.5升锥形瓶
    3. 离心机
    4. 37℃摇床培养箱
    5. 20℃摇床培养箱
    6. 冰水浴(水和冰混合)

  2. 净化 Tt Ago
    1. 法语压力机,超声波仪或等效物对细胞破裂
    2. 离心机

程序

  1. 在大肠杆菌 KRX中表达 Tt
    1. 取10ml 50ml LB培养基在50ml Greiner管中
    2. 加入10μl1000x氯霉素溶液
    3. 加入10μl1,000x链霉素溶液
    4. 加入200μl20%D-葡萄糖溶液
    5. 使用无菌吸头从甘油原液接种培养物
    6.  在37℃下,在振荡培养箱中以180rpm培养o/n
    7. 在o/n温育后,在室温下以4000xg离心培养10分钟。
    8. 除去上清液以去除任何痕量的D-葡萄糖 - 这是 重要的作为D-葡萄糖痕迹可以防止L-鼠李糖吸收(和从而   抑制 Tt Ago表达)。
    9. 重悬细胞沉淀在1ml LB培养基。
    10. 取含有1 L LB培养基的2.5升锥形瓶
    11. 加入1ml 1000x氯霉素溶液
    12. 加入1ml 1,000x链霉素溶液
    13. 添加重悬细胞。
    14. 在37℃下在振荡培养箱中以140rpm孵育培养物
    15. 监测培养物的OD 600nm。 如果OD 600nm 0.7-0.8是 达到(这需要〜3-4小时),将培养物转移到冰水浴中 冷冻15分钟。 这一步骤减缓了E的代谢。 大肠杆菌并触发冷激蛋白的表达,这可能有助于Tt Ago 折叠在表达期间。
    16. 向培养物中加入1ml 1M IPTG和5ml 20%L-鼠李糖溶液以诱导表达。
    17. 将培养物转移到20℃摇床培养箱中,以140rpm转移o/n(+/- 16h)表达
    18. 通过在4℃下以6,000xg离心培养物15分钟收获细胞。
    19. 除去上清液。 你通常最终会有4-5克的细胞沉淀。

  2. 净化 Tt Ago
    1. 如果使用French Press进行细胞裂解:在缓冲液I中重悬细胞沉淀   (每克细胞沉淀5ml缓冲液I),并通过细胞悬浮液 通过法语挤压两次,在16,000psi(见注c)。
      如果使用a   用于细胞裂解的超声器:将细胞沉淀重悬于缓冲液I(每克细胞沉淀3ml缓冲液I),并通过超声处理裂解细胞。 使用   适用于大体积细胞裂解的尖端和声处理方案 悬浮液。
    2. 在4℃下以30,000×g离心30分钟
    3. 将上清液(无细胞提取物)转移到干净的试管中
    4. 准备一个Strep-Tactin琼脂糖柱通过加载500微升的50% 悬浮于1.2ml Bio-Spin层析柱(最终柱 体积为250μl)
    5. 通过用500μl缓冲液I装载柱三次来洗涤柱
    6. 将无细胞提取液装入色谱柱。
    7. 通过用750μl缓冲液I装载其三次来洗涤柱
    8. 通过用750μl缓冲液II装载其三次来洗涤柱
    9. 洗脱蛋白质通过加载250μL缓冲液的列三次   三,收集流经。 Tt Ago洗脱的典型产率 级分对于级分1和3以及在5-10中在0-2.5μM范围内   μM范围。部分2.Tt AgoDM的典型产率在0-2.5μM 范围为级分1和3,在2.5-7.5μM范围内为级分2.
      注意:
      1. 根据实验,MgCl 2 和MnCl 2 浓度可以 降低至0.5mM,或省略。 NaCl浓度可以降低至0.5   M,但是, Ago(尤其是在较高的蛋白质浓度下)更多   在1M NaCl下稳定。 在较低浓度盐(特别是波纹管250 mM),在高于5μM的蛋白质浓度下,Ago不稳定。)
      2. 上述协议描述了无指导的 Tt ago的净化。 如果 指导共纯化是需要的,更换所有缓冲液I在此 实验用缓冲液II。
      3. 使用法式压力传感器或 超声波仪给出或多或少相同的产量 - Tt Ago非常稳定, 很少甚至没有蛋白质将在超声处理期间损失。 记住 然而,超声处理通常不太适合大体积,和   适合于裂解大体积细胞悬浮液的方案 应该应用。

代表数据



图1. Tt Ago(左)和 的考马斯亮蓝染色的12%SDS-PAGE凝胶 > Tt AgoDM(右)纯化样品。 M:Bio-Rad精密加蛋白标记。 标记带大小以kDa表示。 FT:柱流过装载的无细胞提取物的部分。 W1:具有缓冲液I的柱洗级分。W2:具有缓冲液II的柱洗级分。 EL1-EL3:洗脱级分1-3。 蛋白质显示为具有+/- 75kDa大小的带。

食谱

  1. LB培养基(1L)
    10g胰蛋白胨
    5g酵母提取物
    10克NaCl
    用demiwater填充1升
    用NaOH将pH设定为7.5
  2. 缓冲区I
    20mM Tris-HCl(pH8)
    1 M NaCl
    2mM MgCl 2/
  3. 缓冲区II
    20mM Tris-HCl(pH8)
    1 M NaCl
    2mM MnCl 2
  4. 缓冲区III
    20mM Tris-HCl(pH8)
    1 M NaCl
    2mM MnCl 2
    2.5mM生物素 注意:生物素可以用d-Desthiobiotin替换,这将允许柱再生。

致谢

这项工作得到了荷兰科学研究组织(NWO)授予J.v.d.O的资助。 (NWO-TOP,854.10.003)。

参考文献

  1. Swarts,DC,Jore,MM,Westra,ER,Zhu,Y.,Janssen,JH,Snijders,AP,Wang,Y.,Patel,DJ,Berenguer,J.,Brouns,SJand van der Oost, 2014)。 由原核生物Argonaute进行的DNA引导的DNA干扰。 自然 507(7491):258-261。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Swarts, D. C., Jore, M. M. and van der Oost, J. (2014). Expression and Purification of the Thermus thermophilus Argonaute Protein. Bio-protocol 4(19): e1253. DOI: 10.21769/BioProtoc.1253.
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