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Sample Preparation of Telomerase Subunits for Crystallization
用于结晶化的端粒酶亚基样品的制备   

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Abstract

Telomerase is a large ribonucleoprotein complex that replicates the linear chromosome ends in most eukaryotes. Large-scale preparation of the telomerase core components in vitro has long been a big challenge in this field, hindering the understanding of the catalytic mechanism of telomerase, as well as slowing down the development of telomerase inhibitors for cancer therapy. We have successfully developed a protocol for large-scale preparation of the TRBD-CR4/5 complex of the medaka telomerase in vitro, and used this method to study the high-resolution structure of the TRBD-CR4/5 complex by X-ray crystallography. This procedure may be also adapted to purify other protein-RNA complexes for structural studies.

Materials and Reagents

  1. pMAL-C2X vector (New England Biolabs, catalog number: N8076S ) with an insertion of the protease 3C recognition site between MBP and the fusion protein
  2. Escherichia coli (E. coli) strain ScarabXpress T7lac cells (Scarab Genomics, catalog number: C-1709-05K )
  3. LB (Luria-Bertani) medium (BD Bioscience, catalog number: 214906 ) and LB agar (BD Bioscience, catalog number: 244520 )
  4. Ampicillin (AMRESCO, catalog number: 0339 )
  5. Glucose (AMRESCO, catalog number: 0188 )
  6. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (AMRESCO, catalog number: 0487 )
  7. Phenylmethylsulfonyl fluoride (PMSF) (Roche Diagnostics, catalog number: 11359061001 )
  8. Tris(2-carboxyethyl)phosphine (TCEP) (AMRESCO, catalog number: K831 )
  9. Benzamidine (Sigma-Aldrich, catalog number: 12072 )
  10. Leupeptin (Sigma-Aldrich, catalog number: 62070 )
  11. Pepstatin (Sigma-Aldrich, catalog number: 77170 )
  12. Amylose resin (New England Biolabs, catalog number: E8021S )
  13. Ammonium sulfate (Sigma-Aldrich, catalog number: A4418 )
  14. Sodium chloride (Sigma-Aldrich, catalog number: S3014 )
  15. Magnesium Chloride (Sigma-Aldrich, catalog number: 63068 )
  16. Tris-HCl (Amresco, catalog number: 0 497 )
  17. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  18. Maltose (Sigma-Aldrich, catalog number: 1B1184 )
  19. HEPES-KOH (Sigma-Aldrich, catalog number: 54457 )
  20. Spermidine (Sigma-Aldrich, catalog number: 85558 )
  21. Dithiothreitol (DTT) (Roche Diagnostics, catalog number: 10708984001 )
  22. Recombinant RNasin Ribonuclease Inhibitor (Promega Corporation, catalog number: N2511 )
  23. Nuclease-Free Water (Promega Corporation, catalog number: P1193 )
  24. T7 RNA polymerase (New England Biolabs, catalog number: M0251S )
  25. Pyrophosphatase (New England Biolabs, catalog number: M0361S )
  26. Glucosamine-6-phosphoate (Sigma-Aldrich, catalog number: G5509 )
  27. Lysis buffer (see Recipes)
  28. Column buffer (see Recipes)
  29. RNA buffer (see Recipes)
  30. 5x transcription buffer (see Recipes)

Equipment

  1. Incubator shaker (Eppendorf, model: New BrunswickTM Innova 44 )
  2. Centrifuge (Eppendorf, model: 5810R )
  3. Ultracentrifuge (Beckman Coulter, model: Optima TM)
  4. 45Ti rotor (Beckman Coulter)
  5. Sonicator (Branson Sonifier)
  6. AKTA purifier (GE)

Procedure

  1. Preparation of recombinant proteins of the TRBD subunit of telomerase
    1. Medaka TRBD subunit (residues 318-579) was inserted into a modified pMAL-2CX vector and the plasmid was transformed into the E. coli strain ScarabXpress T7lac cells.
    2. Pick a single colony from a freshly streaked plate. Inoculate a starter culture of 5 ml LB medium containing 100 µg/ml ampicillin in a sterile plastic tube. Incubate at 37 °C overnight with shaking at 250 rpm.
    3. Dilute the overnight culture into 2 L LB medium containing 0.2% glucose and 100 µg/ml ampicillin, and grow the culture at 37 °C with shaking at 250 rpm until the cell density reaches OD600 = 0.5.
    4. Induce the expression of the recombinant TRBD proteins with 0.1 mM IPTG at 20 °C for 16 h with shaking at 250 rpm.
    5. Harvest cells by centrifugation at 4,000 rpm for 15 min at 4 °C (Beckman centrifuge).
    6. Resuspend the cell pellet in 50 ml lysis buffer supplemented with 1 mM PMSF, 5 mM benzamidine, 1 µg/ml leupeptin, and 1 µg/ml pepstatin.
    7. Sonicate the cell suspension with 6 short burst of 30 sec followed by intervals of 30 sec for cooling (sonication power 100 W).
    8. Remove cell debris by ultracentrifuge at 4 °C for 40 min at 46,000 rpm using a 45Ti rotor.
    9. Slowly add ammonium sulfate (powder) into cell lysate to 55% saturation (add 16.3 g ammonium sulfate per 50 ml cell lysate). Mix for 30 min at 4 °C with slow stirring.
    10. Pellet the precipitated proteins by centrifuge at 4 °C for 15 min at 15,000 rpm using a 45Ti rotor.
    11. Solubilize the protein pellet in lysis buffer and mix it with 5 ml pre-equilibrated amylose resin in a 50-ml conical tube. Rock the tube for 2 h at 4 °C.
    12. Transfer the bead-protein mixture to a BIO-RAD Econo-Pac column and allow all of the liquid to flow through the column. Then, wash beads with 100 ml lysis buffer.
    13. Elute the MBP-TRBD proteins with 15 ml lysis buffer added with 10 mM maltose.
    14. Apply the eluted MBP-TRBD proteins to a HiLoad Superdex 200 chromatography column to perform gel filtration purification. The column is pre-equilibrated with 120 ml lysis buffer.
    15. Collect the fractions that contain the MBP-TRBD proteins, and treat them with 200 μg protease 3C at 4 °C overnight to cleave the fusion proteins.
    16. Slowly pass the protease 3C-digested protein solution through 5 ml pre-equilibrated amylose resin by gravity to remove MBP and the uncleaved MBP-TRBD proteins.
    17. Apply the TRBD proteins to a HiLoad Superdex 200 chromatography column to perform gel filtration purification. The column is pre-equilibrated with 120 ml column buffer.
    18. Concentrate the purified proteins using Amicon 15 ml centrifugal filter to a concentration of 10 mg/ml (determined by UV280 absorbance) and store them at -80 °C in small aliquots.

  2. Preparation and purification of the CR4/5 RNAs
    1. Prepare the DNA templates for the in vitro transcription of the medaka telomerase CR4/5 RNA (nucleotides 170-220), with a glucosamine-6-phosphate-activated ribozyme (GlmS ribozyme) fused at its 3’ terminus, by PCR amplification.
    2. Set up 5 ml of the following in vitro transcription reaction and carry it out at 37 °C overnight.
      1 ml
      5x transcription buffer
      1 ml
      NTP mix (25 mM of each NTP)
      0.5 ml
      DNA template (200 µg/ml)
      2.375 ml
      Nuclease-free water
      50 µl
      Recombinant RNasin ribonuclease inhibitor
      50 µl
      T7 RNA polymerase (3 mg/ml)
      25 µl
      Pyrophosphatase (100 U/ml)
    3. After the in vitro transcription, purify the CR4/5-GlmS-ribozyme RNAs from the DNA templates and the excess NTPs by gel-filtration chromatography on a HiLoad Superdex 200 column with the RNA buffer.
    4. Pool and concentrate the RNA fractions. Treat them with 1 mM glucosamine-6-phosphate at 25 °C for 30 min to cleave the GlmS ribozyme from the 3’ site of CR4/5.
    5. Run another gel-filtration chromatography on HiLoad Superdex 200 with the column buffer to purify the CR4/5 RNAs from the GlmS ribozyme.
    6. Concentrate the CR4/5 RNAs to 10 mg/ml.

  3. Preparation of the TRBD-CR4/5 complex for crystallization
    1. Mix the TRBD proteins and the CR4/5 RNAs at a molar ratio of 1:2, and incubate the mixture on ice for 1 h.
    2. Purify the TRBD-CR4/5 mixture from the excess CR4/5 RNA by gel-filtration chromatography on a HiLoad Superdex 200 column with the column buffer.
    3. Concentrate the TRBD-CR4/5 protein-RNA complexes to 12 mg/ml. Proceed to crystallization trials or store them at -80 °C in small aliquots.

Representative data



Figure 1. SDS-PAGE of the TRBD proteins after the final-step gel filtration


Figure 2. Gel filtration profile of the final-step purification of the TRBD-CR4/5 complex

Recipes

  1. Lysis buffer
    50 mM
    Tris-HCl (pH 7.5)
    500 mM
    NaCl
    1 mM
    MgCl2
    10% (v/v)
    Glycerol
    1 mM
    Tris(2-carboxyethyl)phosphine (TECP)
  2. Column buffer
    50 mM
    Tris-HCl (pH 7.5)
    500 mM
    NaCl
    1 mM
    MgCl2
    1 mM
    Tris(2-carboxyethyl)phosphine (TECP)
  3. RNA buffer
    50 mM
    HEPES-KOH (pH 7.5)
    150 mM
    NaCl
    10 mM
    MgCl2
  4. 5x transcription buffer
    400 mM
    HEPES-KOH (pH 7.5)
    120 mM
    MgCl2
    10 mM
    spermidine
    200 mM
    DTT

Acknowledgments

M. L. is supported as a Howard Hughes Medical Institute Early Career Scientist. This work was supported by grants from the Ministry of Science and Technology of China (2013CB910400 to M. L.), the National Natural Science Foundation of China (31330040 to M. L.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB08010201 to M. L.) and the US National Institutes of Health (RO1GM094450 to J. J.­L. C.).

References

  1. Bley, C. J., Qi, X., Rand, D. P., Borges, C. R., Nelson, R. W. and Chen, J. J. (2011). RNA-protein binding interface in the telomerase ribonucleoprotein. Proc Natl Acad Sci U S A 108(51): 20333-20338.
  2. Huang, J., Brown, A. F., Wu, J., Xue, J., Bley, C. J., Rand, D. P., Wu, L., Zhang, R., Chen, J. J. and Lei, M. (2014). Structural basis for protein-RNA recognition in telomerase. Nat Struct Mol Biol 21(6): 507-512.

简介

端粒酶是在大多数真核生物中复制线性染色体末端的大核糖核蛋白复合物。 体外大规模制备端粒酶核心组分长期以来一直是该领域的一个大挑战,阻碍了对端粒酶的催化机理的理解,以及减慢端粒酶抑制剂对癌症的发展 治疗。 我们成功地开发了用于大规模制备体外的medaka端粒酶的TRBD-CR4/5复合物的方案,并且使用该方法研究TRBD-CR4/5复合物的高分辨率结构, 5复合物通过X射线晶体学。 该程序也可以适于纯化其它蛋白质-RNA复合物用于结构研究。

材料和试剂

  1. 在MBP和融合蛋白之间插入蛋白酶3C识别位点的pMAL-C2X载体(New England Biolabs,目录号:N8076S)
  2. 大肠杆菌(大肠杆菌)菌株ScarabXpress T7lac细胞(Scarab Genomics,目录号:C-1709-05K)
  3. LB(Luria-Bertani)培养基(BD Bioscience,目录号:214906)和LB琼脂(BD Bioscience,目录号:244520)
  4. 氨苄青霉素(AMRESCO,目录号:0339)
  5. 葡萄糖(AMRESCO,目录号:0188)
  6. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(AMRESCO,目录号:0487)
  7. 苯基甲基磺酰氟(PMSF)(Roche Diagnostics,目录号:11359061001)
  8. 三(2-羧乙基)膦(TCEP)(AMRESCO,目录号:K831)
  9. 苄脒(Sigma-Aldrich,目录号:12072)
  10. 亮肽素(Sigma-Aldrich,目录号:62070)
  11. 胃酶抑素(Sigma-Aldrich,目录号:77170)
  12. 直链淀粉树脂(New England Biolabs,目录号:E8021S)
  13. 硫酸铵(Sigma-Aldrich,目录号:A4418)
  14. 氯化钠(Sigma-Aldrich,目录号:S3014)
  15. 氯化镁(Sigma-Aldrich,目录号:63068)
  16. Tris-HCl(Amresco,目录号:0497)
  17. 甘油(Sigma-Aldrich,目录号:G5516)
  18. 麦芽糖(Sigma-Aldrich,目录号:1B1184)
  19. HEPES-KOH(Sigma-Aldrich,目录号:54457)
  20. 亚精胺(Sigma-Aldrich,目录号:85558)
  21. 二硫苏糖醇(DTT)(Roche Diagnostics,目录号:10708984001)
  22. 重组RNasin核糖核酸酶抑制剂(Promega Corporation,目录号:N2511)
  23. 无核酸酶水(Promega公司,目录号:P1193)
  24. T7 RNA聚合酶(New England Biolabs,目录号:M0251S)
  25. 焦磷酸酶(New England Biolabs,目录号:M0361S)
  26. 葡糖胺-6-磷酸(Sigma-Aldrich,目录号:G5509)
  27. 裂解缓冲液(见配方)
  28. 列缓冲区(参见配方)
  29. RNA缓冲液(参见配方)
  30. 5x转录缓冲液(见配方)

设备

  1. 孵育摇床(Eppendorf,型号:New Brunswick Innova 44)
  2. 离心机(Eppendorf,型号:5810R)
  3. 超速离心机(Beckman Coulter,型号:Optima )
  4. 45Ti转子(Beckman Coulter)
  5. 超声波仪(Branson Sonifier)
  6. AKTA净化器(GE)

程序

  1. 制备端粒酶TRBD亚单位的重组蛋白
    1. 将Medaka TRBD亚基(残基318-579)插入修饰的 pMAL-2CX载体,并将质粒转化到E中。 大肠杆菌菌株 ScarabXpress T7lac细胞
    2. 选择一个殖民地 从新鲜的斑纹板。 接种5ml LB的发酵剂培养物 在无菌塑料管中含有100μg/ml氨苄青霉素的培养基。 在37℃下以250rpm振荡孵育过夜
    3. 稀释 过夜培养到含有0.2%葡萄糖和100μl的2L LB培养基中 μg/ml氨苄青霉素,在37℃,250rpm振荡下培养 直到细胞密度达到OD 600 = 0.5
    4. 诱导重组TRBD蛋白用0.1mM IPTG在20℃下以250rpm振摇16小时表达。
    5. 收获细胞通过在4℃下以4,000rpm离心15分钟(Beckman离心机)
    6. 重悬细胞沉淀在50ml裂解缓冲液中, mM PMSF,5mM苄脒,1μg/ml亮肽素和1μg/ml胃蛋白酶抑制剂。
    7. 超声处理细胞悬液6短暂爆发30秒,然后 以30秒的间隔冷却(超声波功率100W)
    8. 通过超速离心机在4℃下使用45Ti转子以46,000rpm离心细胞碎片40分钟
    9. 将硫酸铵(粉末)缓慢加入细胞裂解液中至55% 饱和(每50ml细胞裂解物加入16.3g硫酸铵)。 混合 缓慢搅拌下在4℃下搅拌30分钟
    10. 通过使用45Ti转子在4℃下以15,000rpm离心15分钟来沉淀沉淀的蛋白质
    11. 溶解蛋白质沉淀在裂解缓冲液中,并与5ml混合 预平衡的直链淀粉树脂在50-ml锥形管中。 摇动管 在4℃下保持2小时
    12. 将珠子 - 蛋白混合物转移到BIO-RAD Econo-Pac柱,并允许所有液体流过柱。   然后,用100ml裂解缓冲液洗涤珠子
    13. 用加入10mM麦芽糖的15ml裂解缓冲液洗脱MBP-TRBD蛋白。
    14. 将洗脱的MBP-TRBD蛋白应用于HiLoad Superdex 200 色谱柱进行凝胶过滤纯化。 列   用120ml裂解缓冲液预平衡
    15. 收集 含有MBP-TRBD蛋白的级分,并用200μg处理它们   蛋白酶3C在4℃过夜以切割融合蛋白。
    16. 将蛋白酶3C-消化的蛋白溶液缓慢通过5ml 预平衡的直链淀粉树脂通过重力去除MBP和 未切割的MBP-TRBD蛋白。
    17. 将TRBD蛋白应用于a HiLoad Superdex 200色谱柱进行凝胶过滤 纯化。 该柱用120ml柱缓冲液预平衡
    18. 使用Amicon 15ml离心浓缩纯化的蛋白质 过滤至浓度为10mg/ml(通过UV 280吸光度测定) 并将它们以小等分试样储存在-80℃。

  2. CR4/5 RNA的制备和纯化
    1. 准备用于体外转录的DNA模板 medaka端粒酶CR4/5RNA(核苷酸170-220),具有a 葡糖胺-6-磷酸激活的核酶(GlmS核酶) 3'末端,通过PCR扩增
    2. 设置5ml以下体外转录反应,并在37℃下过夜。
      1 ml
      5x转录缓冲液
      1 ml
      NTP混合物(25mM的每种NTP)
      0.5 ml
      DNA模板(200μg/ml)
      2.375 ml
      无核酸酶水溶液
      50微升
      重组RNasin核糖核酸酶抑制剂
      50微升
      T7 RNA聚合酶(3mg/ml)
      25μl
      焦磷酸酶(100U/ml)
    3. 在体外转录后,纯化CR4/5-GlmS-核酶 来自DNA模板的RNA和过量的NTP通过凝胶过滤 在具有RNA缓冲液的HiLoad Superdex 200柱上进行色谱。
    4. 合并并浓缩RNA级分。 用1mM处理它们 葡糖胺-6-磷酸在25℃下消化30分钟以切割GlmS核酶 从CR4/5的3'位点开始
    5. 再次进行凝胶过滤 在HiLoad Superdex 200上用柱缓冲液进行色谱纯化 来自GlmS核酶的CR4/5RNA
    6. 将CR4/5 RNA浓缩至10mg/ml

  3. TRBD-CR4/5络合物的结晶制备
    1. 以1:2的摩尔比混合TRBD蛋白和CR4/5RNA,并将混合物在冰上孵育1小时。
    2. 从过量的CR4/5 RNA纯化TRBD-CR4/5混合物 在HiLoad Superdex 200柱上进行凝胶过滤层析 列缓冲区。
    3. 浓缩TRBD-CR4/5蛋白-RNA复合物 至12mg/ml。 进行结晶试验或储存在-80°C 以小等分试样。

代表数据



图1.最终步骤凝胶过滤后的TRBD蛋白的SDS-PAGE


图2. TRBD-CR4/5复合物的最终步骤纯化的凝胶过滤曲线

食谱

  1. 裂解缓冲液
    50 mM
    Tris-HCl(pH 7.5)
    500 mM
    NaCl
    1 mM
    MgCl 2
    10%(v/v)
    甘油
    1 mM
    三(2-羧乙基)膦(TECP)
  2. 列缓冲区
    50 mM
    Tris-HCl(pH 7.5)
    500 mM
    NaCl
    1 mM
    MgCl 2
    1 mM
    三(2-羧乙基)膦(TECP)
  3. RNA缓冲液
    50 mM
    HEPES-KOH(pH7.5)
    150 mM
    NaCl
    10 mM
    MgCl 2
  4. 5x转录缓冲液
    400 mM
    HEPES-KOH(pH7.5)
    120 mM
    MgCl 2
    10 mM
    亚精胺
    200 mM
    DTT

致谢

M. L.被支持作为霍华德休斯医学研究所早期职业科学家。这项工作得到了中国科学技术部(2013CB910400到ML),中国国家自然科学基金(31330040到ML),中国科学院战略优先研究计划(XDB08010201到ML)和美国国家卫生研究院(RO1GM094450至JJLC)。

参考文献

  1. Bley,C.J.,Qi,X.,Rand,D.P.,Borges,C.R.,Nelson,R.W.and Chen,J.J。(2011)。 端粒酶核糖核蛋白中的RNA-蛋白结合界面。美国国家科学院院刊USA 108(51):20333-20338。
  2. Huang,J.,Brown,A. F.,Wu,J.,Xue,J.,Bley,C.J.,Rand,D.P.,Wu,L.,Zhang,R.,Chen,J.J.and Lei, 端粒酶中蛋白-RNA识别的结构基础。Nat Struct Mol Biol 21(6):507-512。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Huang, J., Bley, C. J., Rand, D. P., Chen, J. . and Lei, M. (2015). Sample Preparation of Telomerase Subunits for Crystallization. Bio-protocol 5(16): e1565. DOI: 10.21769/BioProtoc.1565.
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