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Expression, Purification and Enzymatic Assay of Plant Histone Deacetylases
植物组蛋白去乙酰化酶的表达,纯化和酶活测定   

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Abstract

Histone deacetylases (HDACs) catalyzing the removal of acetyl groups from lysine residues of histone and non-histone proteins play vital roles in regulation of gene transcription. In plants, HDACs can be grouped into three families, including RPD3-type, SIR2-type and plant specific HD2-type HDACs. Here we describe a method to determine plant HDAC enzymatic activity. This protocol includes expression, purification and enzymatic activity assay of recombinant plant HDACs expressed in Escherichia coli (E. coli) and Arabidopsis thaliana (A. thaliana).

Keywords: Histone deacetylases(组蛋白脱乙酰基酶类), HDACs(HDACs), Arabidopsis(拟南芥)

Materials and Reagents

  1. Sterile Syringe Filters (Merck Millipore Corporation, Millex, catalog number: SLGV033RS )
  2. Ice
  3. Seeds of A. thaliana ecotype Columbia (Col-0)
  4. Escherichia coli (BL21) (Thermo Fisher Scientific, InvitrogenTM, catalog number: C6000-03 )
  5. pGEX-4T-3 expression vector (GE Healthcare, Amersham, catalog number: 28-9545-52 )
  6. Agrobacterium (GV3101)
  7. LB medium (Caisson Laboratories, catalog number: LBP01-500 GM )
  8. Murashige and Skoog (MS) media (Sigma-Aldrich, catalog number: M5524 )
  9. Ampicillin (Beyotime, catalog number: ST007 )
  10. Isopropyl-b-D-thiogalactopyranoside (IPTG) (Sigma-Aldrich, catalog number: 367-93-1 )
  11. Sucrose (Sigma-Aldrich, catalog number: 57-50-1 )
  12. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 1310-58-3 )
  13. Bacto Agar (Sigma-Aldrich, catalog number: 9002-18-0 )
  14. Ethanol
  15. NP40 (Sigma-Aldrich, Abcam, catalog number: 9016-45-9 )
  16. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434 )
  17. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: 7447-40-7 )
  18. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: 7558-79-4 )
  19. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: 7778-77-0 )
  20. Hydrochloric acid (HCl)
  21. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: 60-00-4 )
  22. Glycerol (Sigma-Aldrich, catalog number: 56-81-5 )
  23. L-Glutathione reduced (Sigma-Aldrich, Abcam, catalog number: 70-18-8 )
  24. Ultrapure water
  25. Liquid N2
  26. GST.BindTM Resin (Merck Millipore Corporation, Novagen, catalog number: 70541 )
  27. Protease inhibitors (Roche Diagnostics, catalog number: 11873580001 )
  28. Glycine (Sigma-Aldrich, catalog number: 15527 )
  29. Tris base (Sigma-Aldrich, catalog number: 77-86-1 )
  30. GFP-Trap (ChromoTek, GFP-Trap®_M)
  31. HDAC activity colorimetric assay kit (BioVision, catalog number: K331-100 )
  32. HeLa nuclear extracts (Biovision, catalog number: 1641-1 )
  33. Trichostatin A (Sigma-Aldrich, catalog number: 58880-19-6 )
  34. Bradford Protein Assay Kit (Beyotime, catalog number: P0006 )
  35. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
  36. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  37. Boc-Lys(Ac)-pNA
  38. Phosphate buffered saline (PBS) (see Recipes)
  39. GST Elution buffer (see Recipes)
  40. Plant protein extraction buffer (see Recipes)
  41. GFP Wash buffer (see Recipes)
  42. GFP Elution buffer (see Recipes)
  43. HDAC Assay Buffer (Biovision) (see Recipes)
  44. HDAC substrate (colorimetric substrate) (see Recipes)

Equipment

  1. Plant growth chamber (Panasonic Healthcare Corporation, model: MLR-352 )
  2. Sterile fume hood (Alibab, Airtech, model: VS-1300L )
  3. Autoclave (HIRAYAMA, model: HVE-50 )
  4. Centrifuge (Eppendorf AG, model: 5418R and 5810R )
  5. Shaker (Eppendorf AG, model: New BrunswickTM Innova® 40 )
  6. Sonicator (SONICS & MATERIALS, model: VCX130 )
  7. Spectrophotometer (Molecular Devices Spectra Max)

Procedure

  1. Expression and purification of recombinant HDACs in E. coli
    1. Insert the protein coding sequence of HDACs (e.g. HDA5) into the vector pGEX-4T-3 as described in Luo et al. (2015).
    2. Transform the plasmid into E. coli (BL21) and select on fresh agar plates containing ampicillin (100 µg/ml).
    3. Inoculate a single colony of E. coli into 2 ml LB media with rotation overnight at 37 °C.
    4. Transfer 2 ml E. coli culture into 250 ml LB media with vigorous aeration at 37 °C and culture until OD600 reached 0.4-0.6.
    5. Add a final concentration of 0.1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) into bacteria solution and incubate with vigorous aeration about 6-10 h at 28 °C.
    6. Centrifuge the cells at 6,000 x g for 10 min at 4 °C and remove the supernatant. Then resuspend the pellet with 25 ml of cell lysis buffer (1x PBS).
    7. Sonicate the resuspended solution for 15 sec with 40% power for 40 times. Between each sonication treatment, place the solution on ice for 1 min.
    8. After sonication, apply the soluble cell extract to GST affinity column.
    9. Wash unbound proteins from the resin by adding 5 ml x 3 of wash buffer (1x PBS).
    10. Elute the bound protein from the resin by adding 4 ml elution buffer.
    11. Store the eluted protein at -20 °C.

  2. Expression and purification of HDAC in transgenic plants
    1. Clone HDAC cDNA (e.g. HDA5) into the pK7WGFP binary vector.
    2. Transform the resultant plasmid into GV3101 Agrobacterium strain by electroporation.
    3. Introduce the transformed Agrobacterium into Arabidopsis plants by floral dip (Zhang et al., 2006).
    4. Select putative transformants on ½ MS media containing kanamycin (50 mg/L).
    5. After screening, extract total soluble proteins by plant protein extraction buffer from transgenic plants, then centrifuge at 4 °C and 13,000 x g for 10 min.
    6. Transfer the suspension to a fresh Eppendorf tube and centrifuge again at 4 °C and 13,000 x g for 10 min.
    7. Purify GFP-HDAC recombinant protein by GFP-Trap according to the manufacturer’s instructions. Add 25 µl bead slurry into soluble protein extract.
    8. Tumble-end-over-end for 1 h at 4 °C.
    9. Magnetically separate beads until supernatant is clear. Discard supernatant and repeat wash twice.
    10. Elute bound proteins by adding 50 µl, 0.2 M glycine (pH 2.5).
    11. Transfer the supernatant to a fresh Eppendorf tube and add 5 µl 1 M Tris base (pH 10.4) for neutralization.
    12. Determine the protein concentration of the purified protein by Bradford assay approach (He, 2011).

  3. HDAC enzymatic activity assay
    1. Place 85 µl purified proteins in the 96-well plate, then add 10 µl of 10x HDAC assay buffer and 5 µl of colorimetric substrate to each well and incubate at 37 °C for 1 h. HeLa nuclear extracts (4 µg) were used as positive controls. The HDAC inhibitor TSA was used to demonstrate the specificity of deacetylation activities.
    2. Stop the reaction by adding 10 µl of Lys developer and incubate the plate at 37 °C for 30 min.
    3. Measure the HDAC activity spectrophotometrically at 405 nm.

Representative data

For representative data, please see the papers of Luo et al. (2015).

Recipes

  1. Phosphate buffered saline (PBS) (sterile filtered)
    10 mM Na2HPO4
    2 mM KH2PO4
    137 mM NaCl
    2.7 mM KCl
    pH 7.4
  2. GST elution buffer (sterile filtered)
    50 mM Tris-HCl
    10 mM reduced glutathione
    pH 8.0
  3. Plant protein extraction buffer (sterile filtered)
    10 mM Tris/Cl (pH 7.5)
    150 mM NaCl
    0.5 mM EDTA
    0.5% NP40
  4. GFP wash buffer (sterile filtered)
    10 mM Tris/Cl (pH 7.5)
    50 mM NaCl
    0.5 mM EDTA
  5. GFP elution buffer (sterile filtered)
    200 mM glycine (pH 2.5)
  6. HDAC assay buffer
    15 mM Tris-HCl (pH 8)
    250 μM EDTA
    250 μM NaCl
    10% glycerol
  7. HDAC substrate
    10 mM Boc-Lys(Ac)-pNA

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31200965) and the China Postdoctoral Science Foundation (2014M562220). The histone deacetylase activity assay was modified from instruction of Biovision HDAC activity colorimetric assay kit.

References

  1. He, F. (2011). Bradford protein assay. Bio-protocol Bio101: e45.
  2. Luo, M., Tai, R., Yu, C. W., Yang, S., Chen, C. Y., Lin, W. D., Schmidt, W. and Wu, K. (2015). Regulation of flowering time by the histone deacetylase HDA5 in Arabidopsis. Plant J 82(6): 925-936.
  3. Zhang, X., Henriques, R., Lin, S. S., Niu, Q. W. and Chua, N. H. (2006). Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc 1(2): 641-646.

简介

组蛋白脱乙酰酶(HDAC)催化从组蛋白和非组蛋白蛋白的赖氨酸残基去除乙酰基在调节基因转录中起重要作用。 在植物中,HDAC可以分为三个家族,包括RPD3型,SIR2型和植物特异性HD2型HDAC。 在这里我们描述了一种确定植物HDAC酶活性的方法。 该方案包括在大肠杆菌(大肠杆菌)和拟南芥()中表达的重组植物HDAC的表达,纯化和酶活性测定 >拟南芥)。

关键字:组蛋白脱乙酰基酶类, HDACs, 拟南芥

材料和试剂

  1. 无菌注射器过滤器(Merck Millipore Corporation,Millex,目录号:SLGV033RS)

  2. 拟南芥生物型Columbia(Col-0)的种子
  3. 大肠杆菌(BL21)(Thermo Fisher Scientific,Invitrogen TM ,目录号:C6000-03)
  4. pGEX-4T-3表达载体(GE Healthcare,Amersham,目录号:28-9545-52)
  5. 农杆菌(GV3101)
  6. LB培养基(Caisson Laboratories,目录号:LBP01-500GM)
  7. Murashige和Skoog(MS)培养基(Sigma-Aldrich,目录号:M5524)
  8. 氨苄青霉素(Beyotime,目录号:ST007)
  9. 异丙基-b-D-硫代吡喃半乳糖苷(IPTG)(Sigma-Aldrich,目录号:367-93-1)
  10. 蔗糖(Sigma-Aldrich,目录号:57-50-1)
  11. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:1310-58-3)
  12. Bacto琼脂(Sigma-Aldrich,目录号:9002-18-0)
  13. 乙醇
  14. NP40(Sigma-Aldrich,Abcam,目录号:9016-45-9)
  15. 氯化钠(NaCl)(Sigma-Aldrich,目录号:31434)
  16. 氯化钾(KCl)(Sigma-Aldrich,目录号:7447-40-7)
  17. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:7558-79-4)
  18. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:7778-77-0)
  19. 盐酸(HCl)
  20. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:60-00-4)
  21. 甘油(Sigma-Aldrich,目录号:56-81-5)
  22. L谷胱甘肽还原(Sigma-Aldrich,Abcam,目录号:70-18-8)
  23. 超纯水
  24. 液体N <2>
  25. GST Bind TM Resin(Merck Millipore Corporation,Novagen,目录号:70541)
  26. 蛋白酶抑制剂(Roche Diagnostics,目录号:11873580001)
  27. 甘氨酸(Sigma-Aldrich,目录号:15527)
  28. Tris碱(Sigma-Aldrich,目录号:77-86-1)
  29. GFP-Trap(ChromoTek,GFP-Trap _M)
  30. HDAC活性比色测定试剂盒(BioVision,目录号:K331-100)
  31. HeLa核提取物(Biovision,目录号:1641-1)
  32. 曲古抑菌素A(Sigma-Aldrich,目录号:58880-19-6)
  33. Bradford蛋白测定试剂盒(Beyotime,目录号:P0006)
  34. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7906)
  35. 苯基甲磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)
  36. Boc-Lys(Ac)-pNA
  37. 磷酸盐缓冲盐水(PBS)(见Recipes)
  38. GST洗脱缓冲液(见配方)
  39. 植物蛋白提取缓冲液(见配方)
  40. GFP洗涤缓冲液(参见配方)
  41. GFP洗脱缓冲液(见配方)
  42. HDAC分析缓冲液(Biovision)(参见配方)
  43. HDAC底物(比色底物)(参见配方)

设备

  1. 植物生长室(Panasonic Healthcare Corporation,型号:MLR-352)
  2. 无菌通风橱(Alibab,Airtech,型号:VS-1300L)
  3. 高压灭菌器(HIRAYAMA,型号:HVE-50)
  4. 离心机(Eppendorf AG,型号:5418R和5810R)
  5. Shaker(Eppendorf AG,型号:New Brunswick Innova 40)
  6. 超声波发生器(SONICS& MATERIALS,型号:VCX130)
  7. 分光光度计(Molecular Devices Spectra Max)

程序

  1. 表达和纯化重组HDACs。大肠杆菌
    1. 按照Luo等人(2015)所述,将HDACs(例如HDA5)的蛋白质编码序列插入载体pGEX-4T-3中。
    2. 将质粒转化为E。大肠杆菌(BL21),并在含有氨苄青霉素(100μg/ml)的新鲜琼脂平板上选择。
    3. 接种单个菌落。大肠杆菌在2ml LB培养基中,在37℃下旋转过夜
    4. 转移2ml E。大肠杆菌培养物在250ml LB培养基中在37℃剧烈通气并培养直到OD 600达到0.4-0.6。
    5. 添加终浓度为0.1mM 异丙基-β-D-硫代吡喃半乳糖苷(IPTG) 在28℃下剧烈通气孵育约6-10小时
    6. 离心机 ?细胞在4℃下6,000×g离心10分钟,并除去上清液。 然后用25ml细胞裂解缓冲液(1x PBS)重悬沉淀
    7. 超声处理重悬的溶液15秒,40%功率为40 次。在每次超声处理之间,将溶液置于冰上1 ?min。
    8. 超声处理后,将可溶性细胞提取物应用于GST亲和柱
    9. 通过加入5ml×3的洗涤缓冲液(1×PBS)从树脂中洗涤未结合的蛋白质
    10. 通过加入4ml洗脱缓冲液从树脂中洗脱结合的蛋白质
    11. 将洗脱的蛋白储存在-20℃。

  2. HDAC在转基因植物中的表达和纯化
    1. 将克隆HDAC cDNA(例如HDA5)克隆到pK7WGFP二元载体中。
    2. 通过电穿孔将所得质粒转化到GV3101土壤杆菌菌株中
    3. 通过花浸渍将转化的土壤杆菌引入拟南芥植物中(Zhang,et al。,,2006)。
    4. 在含有卡那霉素(50mg/L)的1/2MS培养基上选择推定的转化体。
    5. 筛选后,通过植物蛋白提取总可溶性蛋白 提取缓冲液从转基因植物,然后在4℃离心 13,000 x g 10分钟。
    6. 将悬浮液转移到新鲜的Eppendorf管中,并在4℃和13,000×g下再次离心10分钟。
    7. 通过GFP-Trap纯化GFP-HDAC重组蛋白 制造商的说明。加入25μl珠浆液到可溶性蛋白质 提取。
    8. 在4℃下翻滚翻滚1小时。
    9. 磁力分离珠直到上清液澄清。弃去上清液并重复洗涤两次
    10. 通过加入50μl,0.2M甘氨酸(pH2.5)洗脱结合的蛋白质
    11. 将上清液转移到新鲜的Eppendorf管中,加入5μl1M Tris碱(pH 10.4)中和。
    12. 通过Bradford测定法确定纯化蛋白质的蛋白质浓度(He,2011)。

  3. HDAC酶活性测定
    1. 放置85微升纯化的蛋白质在96孔板,然后加入10微升 10x HDAC测定缓冲液和5μl比色底物至每个孔 并在37℃孵育1小时。 HeLa核提取物(4μg)用作 ?阳性对照。使用HDAC抑制剂TSA来证明 脱乙酰活性的特异性
    2. 通过加入10μlLys显色剂停止反应,并将板在37℃孵育30分钟。
    3. 在405nm下用分光光度法测量HDAC活性。

代表数据

有代表性的数据,请参阅Luo等人的论文。(2015)。

食谱

  1. 磷酸盐缓冲盐水(PBS)(无菌过滤)
    10mM Na 2 HPO 4
    2mM KH 2 PO 4 sub/
    137 mM NaCl 2.7 mM KCl
    pH 7.4
  2. GST洗脱缓冲液(无菌过滤)
    50mM Tris-HCl
    10 mM还原型谷胱甘肽
    pH 8.0
  3. 植物蛋白提取缓冲液(无菌过滤)
    10mM Tris/Cl(pH7.5) 150mM NaCl 0.5mM EDTA 0.5%NP40
  4. GFP洗涤缓冲液(无菌过滤)
    10mM Tris/Cl(pH7.5) 50mM NaCl 0.5mM EDTA
  5. GFP洗脱缓冲液(无菌过滤)
    200mM甘氨酸(pH 2.5)
  6. HDAC测定缓冲液
    15mM Tris-HCl(pH8)
    250μMEDTA
    250μMNaCl
    10%甘油
  7. HDAC基材
    10mM Boc-Lys(Ac)-pNA

确认

这项工作得到了中国国家自然科学基金(31200965)和中国博士后科学基金(2014M562220)的支持。组蛋白脱乙酰酶活性测定从Biovision HDAC活性比色测定试剂盒的说明修改。

参考文献

  1. 他,F.(2011)。 Bradford蛋白质测定 生物协议 Bio101:e45。
  2. Luo,M.,Tai,R.,Yu,C.W.,Yang,S.,Chen,C.Y.,Lin,W.D.,Schmidt,W.and Wu,K。 拟南芥中组蛋白脱乙酰酶HDA5对开花时间的调节 a> Plant J 82(6):925-936。
  3. Zhang,X.,Henriques,R.,Lin,S.S.,Niu,Q.W.and Chua,N.H。(2006)。 农杆菌介导的拟南芥的转化使用花浸法。/a> Nat Protoc 1(2):641-646。
  • English
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Luo, M. and Wu, K. (2016). Expression, Purification and Enzymatic Assay of Plant Histone Deacetylases. Bio-protocol 6(7): e1778. DOI: 10.21769/BioProtoc.1778.
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