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本实验方案简略版
Nov 2018

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Extraction and Purification of Laccases from Rice Stems
水稻茎秆酶的提取与纯化   

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Abstract

Laccases are found in cell walls of plants in very low amounts. This protocol provides an efficient method to purify laccases from rice stems. The method involves three steps: 1) Isolation of total protein from rice stems using buffers with high salt concentration to extract protein from cell walls; 2) Purification of laccases using concanavalin-A beads; and, 3) In-gel staining of laccases with 4-hydroxyindole. Concanavalin-A specifically binds to internal or non-reducing terminal α-D-mannosyl and α-D-glucosyl groups found in glycoproteins and glycolipids. Laccases being glycoproteins binds to concanavalin-A during purification process and eluted with mannose.

Keywords: Laccase (漆酶), Lignin (木质素), Cell wall (细胞壁), Phenylpropanoid pathway (苯丙烷途径), Rice (水稻), Plants (植物)

Background

Laccases are oxidases ubiquitously present in bacteria, fungi, animals, and plants. They are some of the oldest enzymes identified. Laccase is involved in diverse functions such as pigmentation of fungal spores, regeneration of plants, as fungal virulence factors, and in lignification of cell walls and delignification during wood rotting. They oxidize the biosynthesis of secondary metabolite called lignin in vascular tissues of plants. Purification of laccases is very challenging as laccases are expressed in very low amounts. Existing protocols to purify laccases are from either microorganism or softer tissues of plants such as leaves. This protocol provides an efficient approach to extract and purify laccases from harder tissues of plants such as rice stems.

Materials and Reagents

  1. Pipette tips (Tarsons)
  2. Spatula (Fisher Scientific, catalog number: 11533462)
  3. Filter paper (Whatman filters, No. 1)
  4. 15 ml and 50 ml centrifuge tubes (Tarsons)
  5. Dialysis membrane (Thermo Fisher Scientific, 10 kDa molecular weight cut-off (MWCO); catalog number: 68100)
  6. Econo-column glass chromatography columns (Bio-Rad, catalog number: 7371512)
  7. O. sativa var. Pusa Basmati-1 (PB-1)
    Note: PB-1 is an aromatic variety of domesticated rice.
  8. Liquid nitrogen
  9. Concanavalin-A beads (Sigma-Aldrich, catalog number: C7555-5ML)
  10. Sodium chloride (NaCl) (HiMedia, catalog number: MB023-1Kg)
  11. Magnesium chloride (MgCl2) (Fischer Scientific, catalog number: 15535-500G)
  12. Manganese (II) chloride (MnCl2) (Sigma, catalog number: M8054)
  13. Calcium chloride (CaCl2) (Sigma, catalog number: C3306-250G)
  14. Tris-base (Fisher Scientific, catalog number: 15965-500G)
  15. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: 29505)
  16. Tris-HCl (pH 6.8 and 8.8) (Bio-Rad)
  17. Sodium acetate (CH3COONa) (HiMedia, catalog number: MB048-500G)
  18. Polyvinylpyrrolidone (PVP) (Sigma, catalog number: P5288-100G)
  19. Phenylmethylsulfonyl fluoride (PMSF) (Sigma, catalog number: P7626-5G)
  20. Dithiothreitol (DTT) (Roche, catalog number: 11096176001)
  21. Glycine (Sigma, catalog number: G7126-100G)
  22. Protein cocktail inhibitor (Sigma-Aldrich, catalog number: 5056489001)
  23. 4-hydroxyindole (Sigma-Aldrich, catalog number: 219878)
  24. Mannose (Sigma-Aldrich, catalog number: 112585)
  25. Protein concentrator (Sartorius, Vivaspin, 30,000 d MWCO, catalog number: VS15T21)
  26. 30% acrylamide (Bio-Rad, catalog number: 161-0156)
  27. 10% sodium dodecyl sulfate (SDS) (Invitrogen, catalog number: 15525-017)
  28. 10% ammonium persulfate (Sigma, catalog number: 161-0700)
  29. N,N,N’,N’-Tetramethylethylenediamine (TEMED) (Sigma, catalog number: T7024-25ml)
  30. Coomassie brilliant blue (CBB-R-250) (Sigma, catalog number: B-7920-10G)
  31. Bromophenol blue (Sigma, catalog number: B5525-5G)
  32. Glycerol (Millipore, catalog number: DC4P640148)
  33. Ethanol (Emsure, catalog number: 1.00983.0511)
  34. Acetic acid (Fisher Scientific, catalog number: 11005)
  35. Methanol (Merck, catalog number: 60600905001730)
  36. Isopropanol (Fisher Scientific, catalog number: 13825)
  37. Dialysis buffer (see Recipes)
  38. Extraction buffer (see Recipes)
  39. Equilibration buffer (see Recipes)
  40. Wash buffer (see Recipes)
  41. Regeneration buffer (see Recipes)
  42. Storage buffer (see Recipes)
  43. 50 mM Mannose (see Recipes)
  44. 12% SDS-PAGE gel (see Recipes)
  45. TGS buffer (see Recipes)
  46. Native-PAGE gel (see Recipes)
  47. Native gel sample loading dye (6x) (see Recipes)
  48. TG buffer (see Recipes)
  49. CBB-R-250 staining solution (see Recipes)
  50. Destaining solution (see Recipes)
  51. 5 mM 4-hydroxyindole (see Recipes)

Equipment

  1. Scissors
  2. Spatula
  3. Conical flask
  4. Mortar and pestle (Sigma, catalog numbers: Z247472 and Z247510)
  5. Pipettes (Gilson, models: P1000, P200, P100, P20 and P10)
  6. Cold centrifuge (Thermo Scientific, Heraeus Megafuge 16R, 75003694)
  7. -20 °C refrigerator (Panasonic)
  8. Thermomixer (Eppendorf, Thermomixer comfort)
  9. Chemical fume hood
  10. Rocker 
  11. Magnetic stirrer
  12. Magnetic beads
  13. Peristaltic pump (Cole Parmer 7520-67 Masterflex Console Drive, model: 77200-52)

Note: The materials, reagents and equipment not provided with company and catalog number can be ordered from any qualified company for using in this experiment.

Procedure

  1. Extraction of total protein from rice stems
    1. Chill the mortar and pestle with liquid nitrogen and divide 30 g of fresh or -80 °C frozen stem samples into 4 parts of 7.5 g each for convenience and freeze in liquid nitrogen. Take each part of sample, cut the tissue into small pieces of about 1-2 cm long with pre-chilled scissors in a pre-chilled mortar.
    2. Grind the tissue to a fine powder by using mortar and pestle using liquid nitrogen. Collect the powder in a 50 ml centrifuge tube with the help of an ice-chilled spatula and place it in liquid nitrogen. Repeat the same for the remaining sample.
    3. Add 45 ml of extraction buffer to each centrifuge tube and invert the tubes to homogenize. Place the centrifuge tubes on a rocker at 4 °C for 1 h with a set speed of 50 rockings per min. 
    4. Centrifuge for 45 min at 7,177 x g, 4 °C.
    5. Filter the supernatant through Whatman filter paper to a fresh autoclaved conical flask.
    6. Transfer the filtrate into 10 cm long dialysis bags and dialyze overnight in 3 L of dialysis buffer at 4 °C on a stirrer at 120 rpm.
    7. The dialyzed protein sample is collected and used for purification of laccases.

  2. Purification of laccases from crude protein extract
    1. Slowly load the econo-column with concanavalin-A beads using a cut 1 ml pipette tip. Allowed it to settle at the base of the column. Length of separation column was 1.5 cm x 2.5 cm (diameter x length).
    2. Wash the column with 4 column volume (CV) of wash buffer. A flow rate of 1 ml/min was maintained throughout the procedure using a peristaltic pump.
    3. Pre-equilibrate the column with 4 CV of equilibration buffer.
    4. Pass the total crude protein sample through the walls of column slowly without disturbing using a 1 ml pipette.
    5. Wash the column with 3 CV of wash buffer to wash the unbound protein.
    6. Elute laccases with 9 ml of 50 mM mannose in three elutions of 3 ml each.
    7. Load the protein concentrators with purified laccase and centrifuge at 7,177 x g, 4 °C to concentrate the eluted protein to up to 200 μl.
    8. Wash the column with 3 CV of equilibration buffer and then with Milli-Q-filtered water.
    9. Regenerate the column by passing regeneration buffer A and B alternatively for 4 times.
    10. Pass 1 CV of storage buffer through the column and store the beads in storage buffer at 4 °C.
    11. Obtained purified laccases can be used for in-gel staining (Swetha et al., 2018). Perform standard mass-spectrometry (LC-MS) using cut prominent bands around 50-65 kDa from native gel to confirm the presence of laccases and to rule out contamination from peroxidases (as plant peroxidases range from 40-50 kDa).
    12. Load concentrated protein in about 30 μl onto 12% SDS gel and subject to electrophoresis in TGS buffer for 2 h at 80 V.
    13. Stain the gel with CBB overnight and then destain for 4 h in destaining solution at room temperature.

  3. In-gel staining of purified laccases
    1. Sample preparation was done by adding 8 μl of native gel sample loading dye (6x) to the concentrated protein of about 30 μl.
    2. Load the protein sample onto a 12% native gel and subject to electrophoresis in TG buffer for 2 h at 80 V and 4 °C.
    3. Wash the gel with water 3 to 4 times and then stain the gel with 30 ml of 5 mM 4-hydroxyindole for 2 h.

Data analysis

Data analysis could refer to Figures 3 and 4, and also Supplementary figures S7 and S8 from Swetha et al., 2018.

Notes

  1. All the buffers required for protein extraction and purification should be pre-cooled to 4 °C before use.
  2. While making the extraction buffer, first add Tris and salts, cool the buffer to 4 °C and then adjust the pH. Add PMSF and protein cocktail inhibitor to the cooled buffer at the end.
  3. To regenerate the concanavalin column, pass regeneration buffers A and B alternatively starting with buffer A and also ending with buffer A.
  4. Mass spectrometry (LC-MS) has to be employed to confirm the presence of laccases after purification.

Recipes

  1. Dialysis Buffer (3 L)
    100 mM CH3COONa (40.8 g)
    Add Milli-Q-filtered water to a volume of 2.5 L
    Cool to 4 °C and then adjust pH to 4.5 with glacial acetic acid
    Mix and make up the volume to 3 L and store at 4 °C
  2. Extraction buffer (500 ml)
    50 mM Tris (3.025 g)
    1 M NaCl (30 g)
    1.5 M CaCl2 (110.25 g)
    Add Milli-Q-filtered water to a volume of 400 ml
    Cool to 4 °C and then adjust pH to 8 with 1 N HCl
    150 mg DTT
    0.1% (w/v) PVP (0.5 g)
    0.5 M PMSF (87 mg) (first dissolve in 1.5 ml of ethanol and then add to the buffer)
    1 tablet of protein cocktail inhibitor
    Mix and make up the volume to 500 ml and store at 4 °C
    Note: This buffer should be freshly prepared each time.
  3. Equilibration buffer (750 ml)
    50 mM Tris (4.533g)
    Add Milli-Q-filtered water to a volume of 650 ml
    Cool to 4 °C and then adjust pH to 7.5 with 1 N HCl
    0.5 M NaCl (22.5 g)
    1 mM MgCl2 (152.25 mg)
    1 mM CaCl2 (110.25 mg)
    1 mM MnCl2 (148.44 mg)
    Mix and make up the volume to 750 ml and store at 4 °C
  4. Wash buffer (500 ml)
    1 M NaCl (29.22 g)
    5 mM MgCl2 (238 mg)
    5 mM CaCl2 (367.5 mg)
    5 mM MnCl2 (491.6 mg)
    Add Milli-Q-filtered water to a volume of 500 ml and store at 4 °C
  5. Regeneration buffers (each 500 ml)
    1. Regeneration buffer A
      0.1 M Tris (6.057 g)
      Add Milli-Q-filtered water to a volume of 400 ml
      Cool to 4 °C and then adjust pH to 8.5 with 1 N HCl
      0.5 M NaCl (15 g)
      Mix and make up the volume to 500 ml and store at 4 °C
    2. Regeneration buffer B
      0.1 M CH3COONa (6.8 g)
      Add Milli-Q-filtered water to a volume of 400 ml
      Cool to 4 °C and then adjust pH to 4.5 with glacial acetic acid
      1 M NaCl (29.21 g)
      Mix and make up the volume to 500 ml and store at 4 °C
  6. Storage buffer (40 ml)
    0.1 M CH3COONa (544 mg)
    0.1 M NaCl (2.33 g)
    1 mM CaCl2 (5.88 mg)
    Add Milli-Q-filtered water to a volume of 30 ml
    20% ethanol (8 ml)
    Mix and make up the volume to 40 ml, store at 4 °C
  7. 50 mM Mannose (20 ml)
    180 mg of Mannose in 20 ml equilibration buffer
  8. 12% SDS-PAGE gel
    1. Resolving gel buffer (8 ml)
      3.2 ml 30% acrylamide
      2.64 ml Milli-Q-filtered water
      2 ml 1.5 mM Tris-HCl (Bio-Rad) (pH 8.8)
      0.08 ml 10% SDS
      0.08 ml 10% ammonium persulfate
      0.008 ml TEMED
      Note: After pouring stacking gel into the cast, add 500 μl of isopropanol on top.
    2. Stacking gel buffer (5 ml)
      0.83 ml 30% acrylamide
      3.44 ml Milli-Q-filtered water
      0.63 ml 1.5 mM Tris-HCl (Bio-Rad) (pH 6.8)
      0.05 ml 10% SDS
      0.05 ml 10% ammonium persulfate
      0.005 ml TEMED
      Note: Before pouring stacking gel, remove isopropanol, rinse with water and dry by inserting Whatman paper pieces.
  9. TGS buffer (1.5 L, 1x working)
    25 mM Tris (4.5 g)
    192 mM Glycine (21 g)
    0.1% SDS (1.5 g)
    Add Milli-Q-filtered water, mix and make up the volume to 40 ml. Store at room temperature
  10. Native-PAGE gel
    1. Resolving gel buffer (10 ml)
      4 ml 30% acrylamide
      3.3 ml Milli-Q-filtered water
      2.5 ml 1.5 mM Tris-HCl (Bio-Rad), pH 8.8
      0.1 ml 10% ammonium persulfate
      0.01 ml TEMED
      Note: After pouring stacking gel into the cast, add 500 μl of isopropanol on top.
    2. Stacking gel buffer (5 ml)
      0.83 ml 30% acrylamide
      3.44 ml Milli-Q-filtered water
      0.63 ml 1.5 mM Tris-HCl (Bio-Rad), pH 6.8
      0.05 ml 10% ammonium persulfate
      0.005 ml TEMED
      Note: Before pouring stacking gel, remove isopropanol, rinse with water and remove water thoroughly. Pre-run the gel at 4 °C for 30 min before loading the samples.
  11. Native gel sample loading dye (6x)
    187.5 mM Tris-HCl, pH 6.8 with 25% glycerol and 1% bromophenol blue
  12. TG buffer (pH 8.3, 1.5 L, 1x working)
    25 mM Tris (4.5 g)
    192 mM Glycine (21 g)
    Add Milli-Q-filtered water, mix and make up the volume to 1,500 ml and store at 4 °C
  13. CBB-R staining solution (100 ml)
    0.25 g CBB R-250
    45 ml Methanol
    45 ml double-distilled (dd) H2O
    10 ml acetic acid
  14. Destaining solution
    Ethanol:acetic acid:ddH2O = 1:1:2
  15. 5 mM 4-hydroxyindole
    First, dissolve 133.2 mg of 4-hydroxyindole in 2 ml of ethanol (500 mM 4-hydroxyindole). Then take 300 μl of 500 mM in 30 ml of Milli-Q-filtered water to dilute it to 5 mM 4-hydroxyindole

Acknowledgments

This protocol was developed by modifying the method from Jaiswal et al. (2015). Thanks to Prof. K. Veluthambi for rice seeds. The authors acknowledge financial support from Ramanujan Fellowship (SR/S2/RJN-109/2012; Department of Science and Technology, Government of India) to PVS. PVS lab is supported by NCBS-TIFR core funding and a grant (BT/PR12394/AGIII/103/891/2014) from Department of Biotechnology, Government of India. SC acknowledges a fellowship from DBT, India.

Competing interests

The authors declare no competing interests.

References

  1. Jaiswal, N., Pandey, V. P. and Dwivedi, U. N. (2015). Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography. Int J Biol Macromol 72: 326-332.
  2. Swetha, C., Basu, D., Pachamuthu, K., Tirumalai, V., Nair, A., Prasad, M. and Shivaprasad, P. V. (2018). Major domestication-related phenotypes in Indica rice are due to loss of miRNA-mediated laccase silencing. Plant Cell 30(11): 2649-2662.

简介

漆酶以非常低的量存在于植物的细胞壁中。 该方案提供了从稻茎中纯化漆酶的有效方法。 该方法包括三个步骤:1)使用高盐浓度的缓冲液从水稻茎中分离总蛋白,从细胞壁中提取蛋白质; 2)使用伴刀豆球蛋白-A珠子纯化漆酶; 3)漆酶与4-羟基吲哚的凝胶内染色。 伴刀豆球蛋白-A特异性结合糖蛋白和糖脂中发现的内部或非还原性末端α-D-甘露糖基和α-D-葡糖基。 作为糖蛋白的漆酶在纯化过程中与伴刀豆球蛋白-A结合并用甘露糖洗脱。
【背景】漆酶是普遍存在于细菌,真菌,动物和植物中的氧化酶。 它们是一些最古老的酶。 漆酶参与多种功能,例如真菌孢子的色素沉着,植物的再生,真菌毒力因子,以及木材腐烂期间细胞壁的木质化和脱木素。 它们氧化植物维管组织中称为木质素的次生代谢物的生物合成。 漆酶的纯化非常具有挑战性,因为漆酶以非常低的量表达。 纯化漆酶的现有方案来自植物如叶子的微生物或较软组织。 该方案提供了从植物(例如水稻茎)的较硬组织中提取和纯化漆酶的有效方法。

关键字:漆酶, 木质素, 细胞壁, 苯丙烷途径, 水稻, 植物

材料和试剂

  1. 移液器吸头(Tarsons)
  2. Spatula(Fisher Scientific,目录号:11533462)
  3. 滤纸(Whatman过滤器,第1号)
  4. 15毫升和50毫升离心管(塔森)
  5. 透析膜(Thermo Fisher Scientific,截留分子量10 kDa(MWCO);目录号:68100)
  6. Econo-column玻璃色谱柱(Bio-Rad,目录号:7371512)
  7. 0。苜蓿 var。 Pusa Basmati-1(PB-1)
    注意:PB-1是一种芳香品种的驯化米。
  8. 液氮
  9. Concanavalin-A beads(Sigma-Aldrich,目录号:C7555-5ML)
  10. 氯化钠(NaCl)(HiMedia,目录号:MB023-1Kg)
  11. 氯化镁(MgCl 2 )(Fischer Scientific,目录号:15535-500G)
  12. 氯化锰(II)(MnCl 2 )(Sigma,目录号:M8054)
  13. 氯化钙(CaCl 2 )(Sigma,目录号:C3306-250G)
  14. Tris-base(Fisher Scientific,目录号:15965-500G)
  15. 盐酸(HCl)(Fisher Scientific,目录号:29505)
  16. Tris-HCl(pH 6.8和8.8)(Bio-Rad)
  17. 醋酸钠(CH 3 COONa)(HiMedia,目录号:MB048-500G)
  18. 聚乙烯吡咯烷酮(PVP)(Sigma,目录号:P5288-100G)
  19. 苯基甲基磺酰氟(PMSF)(Sigma,目录号:P7626-5G)
  20. 二硫苏糖醇(DTT)(罗氏,目录号:11096176001)
  21. 甘氨酸(Sigma,目录号:G7126-100G)
  22. 蛋白质鸡尾酒抑制剂(Sigma-Aldrich,目录号:5056489001)
  23. 4-羟基吲哚(Sigma-Aldrich,目录号:219878)
  24. 甘露糖(Sigma-Aldrich,目录号:112585)
  25. 蛋白质浓缩器(Sartorius,Vivaspin,30,000 d MWCO,目录号:VS15T21)
  26. 30%丙烯酰胺(Bio-Rad,目录号:161-0156)
  27. 10%十二烷基硫酸钠(SDS)(Invitrogen,目录号:15525-017)
  28. 10%过硫酸铵(Sigma,目录号:161-0700)
  29. N,N,N',N'-四甲基乙二胺(TEMED)(Sigma,目录号:T7024-25ml)
  30. 考马斯亮蓝(CBB-R-250)(Sigma,目录号:B-7920-10G)
  31. 溴酚蓝(Sigma,目录号:B5525-5G)
  32. 甘油(Millipore,目录号:DC4P640148)
  33. 乙醇(Emsure,目录号:1.00983.0511)
  34. 乙酸(Fisher Scientific,目录号:11005)
  35. 甲醇(默克,目录号:60600905001730)
  36. 异丙醇(Fisher Scientific,目录号:13825)
  37. 透析缓冲液(见食谱)
  38. 提取缓冲液(见食谱)
  39. 平衡缓冲液(见食谱)
  40. 洗涤缓冲液(见食谱)
  41. 再生缓冲液(见食谱)
  42. 存储缓冲区(参见食谱)
  43. 50 mM甘露糖(见食谱)
  44. 12%SDS-PAGE凝胶(见食谱)
  45. TGS缓冲区(见食谱)
  46. Native-PAGE凝胶(见食谱)
  47. 天然凝胶样品上样染料(6x)(见食谱)
  48. TG缓冲液(见食谱)
  49. CBB-R-250染色液(见食谱)
  50. Destaining解决方案(见食谱)
  51. 5 mM 4-羟基吲哚(见食谱)

设备

  1. 剪刀
  2. 抹刀
  3. 锥形瓶
  4. 砂浆和杵(Sigma,目录号:Z247472和Z247510)
  5. 移液器(Gilson,型号:P1000,P200,P100,P20和P10)
  6. 冷离心机(Thermo Scientific,Heraeus Megafuge 16R,75003694)
  7. -20°C冰箱(Panasonic)
  8. Thermomixer(Eppendorf,Thermomixer comfort)
  9. 化学通风橱
  10. 摇臂 
  11. 电磁搅拌机
  12. 磁珠
  13. 蠕动泵(Cole Parmer 7520-67 Masterflex控制台驱动器,型号:77200-52)
注意:未在公司和产品目录中提供的材料,试剂和设备可以从任何合格的公司订购,以便在本实验中使用。

程序

  1. 从水稻茎中提取总蛋白
    1. 用液氮冷却研钵和研杵,并将30g新鲜或-80℃冷冻干燥样品分成4份,每份7.5g,以方便并在液氮中冷冻。取出每份样品,用预先冷却的剪刀在预先冷却的砂浆中将组织切成约1-2cm长的小块。
    2. 使用研钵和研杵使用液氮将组织研磨成细粉。在冰冷的刮刀的帮助下将粉末收集在50ml离心管中,并将其置于液氮中。对剩余的样品重复相同的操作。
    3. 向每个离心管中加入45ml提取缓冲液并倒置管以均化。将离心管放在4°C的摇臂上1小时,设定速度为每分钟50次摇摆。 
    4. 在7,177 x g ,4℃下离心45分钟。
    5. 通过Whatman滤纸将上清液过滤至新鲜的高压灭菌的锥形瓶中。
    6. 将滤液转移到10cm长的透析袋中,并在4L透析缓冲液中在4℃下在搅拌器上以120rpm透析过夜。
    7. 收集透析的蛋白质样品并用于漆酶的纯化。

  2. 从粗蛋白质提取物中纯化漆酶
    1. 使用1毫升切割的移液管尖端,用伴刀豆球蛋白A珠缓慢加载经济柱。允许它在柱子底部沉降。分离柱长度为1.5cm×2.5cm(直径×长度)。
    2. 用4柱体积(CV)的洗涤缓冲液洗涤柱子。使用蠕动泵在整个过程中保持1ml / min的流速。
    3. 用4 CV的平衡缓冲液预平衡柱子。
    4. 将总粗蛋白质样品缓慢地通过柱壁,不使用1ml移液管干扰。
    5. 用3 CV的洗涤缓冲液洗涤柱子以洗涤未结合的蛋白质。
    6. 洗脱漆含9ml 50mM甘露糖,三次洗脱,每次3ml。
    7. 用纯化的漆酶装载蛋白质浓缩器并在7,177 x g ,4℃下离心以将洗脱的蛋白质浓缩至最多200μl。
    8. 用3 CV的平衡缓冲液洗涤柱子,然后用Milli-Q过滤的水洗涤。
    9. 通过将再生缓冲液A和B交替传递4次来重新生成色谱柱。
    10. 将1 CV的储存缓冲液通过色谱柱,并将珠子储存在4°C的储存缓冲液中。
    11. 获得的纯化漆酶可用于凝胶内染色(Swetha等,等,2018)。使用来自天然凝胶的约50-65kDa的切割显着条带进行标准质谱(LC-MS)以确认漆酶的存在并排除过氧化物酶的污染(因为植物过氧化物酶的范围为40-50kDa)。
    12. 将浓缩的蛋白质以约30μl加载到12%SDS凝胶上,并在TGS缓冲液中在80V下电泳2小时。
    13. 用CBB将凝胶染色过夜,然后在室温下在脱色溶液中脱色4小时。

  3. 纯化漆酶的凝胶内染色
    1. 通过将8μl天然凝胶样品上样染料(6x)添加至约30μl的浓缩蛋白质来进行样品制备。
    2. 将蛋白质样品加载到12%天然凝胶上,并在80℃和4℃下在TG缓冲液中电泳2小时。
    3. 用水洗涤凝胶3至4次,然后用30ml 5mM 4-羟基吲哚染色凝胶2小时。

数据分析

数据分析可以参考图3和4,以及来自Swetha 等人的补充图S7和S8,,2018。

笔记

  1. 蛋白质提取和纯化所需的所有缓冲液应在使用前预先冷却至4°C。
  2. 在制备提取缓冲液时,首先加入Tris和盐,将缓冲液冷却至4°C,然后调节pH值。最后将PMSF和蛋白质混合抑制剂加入冷却的缓冲液中。
  3. 要重新生成伴刀豆球柱,通过再生缓冲液A和B,或者从缓冲液A开始,也以缓冲液A结束。
  4. 必须使用质谱(LC-MS)来确认纯化后漆酶的存在。

食谱

  1. 透析缓冲液(3升)
    100mM CH 3 COONa(40.8g)
    将Milli-Q过滤水加入2.5L的体积中 冷却至4°C,然后用冰醋酸将pH调节至4.5 将体积混合并补足至3L,并在4°C下储存
  2. 提取缓冲液(500毫升)
    50mM Tris(3.025g)
    1 M NaCl(30 g)
    1.5M CaCl 2 (110.25g)
    将Milli-Q过滤水加入400 ml的体积中 冷却至4°C,然后用1N HCl将pH调节至8 150毫克DTT
    0.1%(w / v)PVP(0.5g)
    0.5M PMSF(87mg)(首先溶于1.5ml乙醇中,然后加入缓冲液中)
    1片蛋白质鸡尾酒抑制剂
    混合并补足体积至500毫升,储存在4°C
    注意:每次都应该为这个缓冲区做好新的准备。
  3. 平衡缓冲液(750毫升)
    50 mM Tris(4.533g)
    将Milli-Q过滤水加入650 ml的体积中 冷却至4°C,然后用1N HCl将pH调节至7.5
    0.5 M NaCl(22.5 g)
    1mM MgCl 2 (152.25mg)
    1mM CaCl 2 (110.25mg)
    1mM MnCl 2 (148.44mg)
    混合并补足体积至750毫升,储存在4°C
  4. 洗涤缓冲液(500毫升)
    1 M NaCl(29.22 g)
    5mM MgCl 2 (238mg)
    5mM CaCl 2 (367.5mg)
    5mM MnCl 2 (491.6mg)
    加入Milli-Q过滤水至500 ml的体积并储存在4°C
  5. 再生缓冲液(每500毫升)
    1. 再生缓冲液A
      0.1M Tris(6.057g)
      将Milli-Q过滤水加入400 ml的体积中 冷却至4°C,然后用1N HCl将pH调节至8.5
      0.5 M NaCl(15 g)
      混合并补足体积至500毫升,并在4°C下储存
    2. 再生缓冲液B
      0.1M CH 3 COONa(6.8g)
      将Milli-Q过滤水加入400 ml的体积中 冷却至4°C,然后用冰醋酸将pH调节至4.5 1 M NaCl(29.21 g)
      混合并补足体积至500毫升,并在4°C下储存
  6. 储存缓冲液(40毫升)
    0.1 M CH 3 COONa(544 mg)
    0.1 M NaCl(2.33 g)
    1mM CaCl 2 (5.88mg)
    加入Milli-Q过滤水至30 ml的体积 20%乙醇(8毫升)
    混合并补足体积至40毫升,储存在4°C
  7. 50 mM甘露糖(20 ml)
    在20ml平衡缓冲液中加入180mg甘露糖
  8. 12%SDS-PAGE凝胶
    1. 分离凝胶缓冲液(8毫升)
      3.2毫升30%丙烯酰胺
      2.64毫升Milli-Q过滤水
      2ml 1.5mM Tris-HCl(Bio-Rad)(pH8.8)
      0.08毫升10%SDS
      0.08毫升10%过硫酸铵
      0.008毫升TEMED
      注意:将堆积凝胶倒入铸件后,在顶部加入500μl异丙醇。
    2. 堆积凝胶缓冲液(5毫升)
      0.83毫升30%丙烯酰胺
      3.44毫升Milli-Q过滤水
      0.63ml 1.5mM Tris-HCl(Bio-Rad)(pH6.8)
      0.05毫升10%SDS
      0.05毫升10%过硫酸铵
      0.005毫升TEMED
      注意:在倒入堆积凝胶之前,取出异丙醇,用水冲洗并插入Whatman纸片干燥。
  9. TGS缓冲液(1.5升,1倍工作)
    25mM Tris(4.5g)
    192 mM甘氨酸(21 g)
    0.1%SDS(1.5克)
    加入Milli-Q过滤水,混合并补足至40 ml的体积。在室温下储存。
  10. Native-PAGE凝胶
    1. 分离凝胶缓冲液(10毫升)
      4毫升30%丙烯酰胺
      3.3毫升Milli-Q过滤水
      2.5ml 1.5mM Tris-HCl(Bio-Rad),pH8.8
      0.1毫升10%过硫酸铵
      0.01毫升TEMED
      注意:将堆积凝胶倒入铸件后,在顶部加入500μl异丙醇。
    2. 堆积凝胶缓冲液(5毫升)
      0.83毫升30%丙烯酰胺
      3.44毫升Milli-Q过滤水
      0.63ml 1.5mM Tris-HCl(Bio-Rad),pH 6.8
      0.05毫升10%过硫酸铵
      0.005毫升TEMED
      注意:在倒入堆积凝胶之前,取出异丙醇,用水冲洗并彻底去除水分。在加载样品之前,将凝胶在4°C下预运行30分钟。
  11. 天然凝胶样品上样染料(6x)
    187.5mM Tris-HCl,pH 6.8,含25%甘油和1%溴酚蓝
  12. TG缓冲液(pH 8.3,1.5 L,1x工作)
    25mM Tris(4.5g)
    192 mM甘氨酸(21 g)
    加入Milli-Q过滤水,混合并补足至1,500 ml的体积,并在4°C下储存。
  13. CBB-R染色溶液(100毫升)
    0.25克CBB R-250
    45毫升甲醇
    45毫升双蒸馏(dd)H 2 O
    10毫升乙酸
  14. Destaining解决方案
    乙醇:乙酸:ddH 2 O = 1:1:2
  15. 5mM 4-羟基吲哚
    首先,将133.2mg 4-羟基吲哚溶于2ml乙醇(500mM 4-羟基吲哚)中。然后在30 ml Milli-Q过滤水中加入300μl500mM稀释至5 mM 4-羟基吲哚。

致谢

该协议是通过修改Jaiswal et al 的方法开发的。 (2015年)。感谢K. Veluthambi教授的水稻种子。作者感谢Ramanujan奖学金(SR / S2 / RJN-109/2012;印度政府科学技术部)对PVS的财政支持。 PVS实验室得到了NCBS-TIFR核心资金和印度政府生物技术部的资助(BT / PR12394 / AGIII / 103/891 / 2014)的支持。 SC承认印度DBT的奖学金。

利益争夺

作者宣称没有竞争利益。

参考

  1. Jaiswal,N.,Pandey,V。P.和Dwivedi,U。N.(2015)。 使用亲和力从番木瓜( Carica papaya )中纯化耐热碱性漆酶色谱法。 Int J Biol Macromol 72:326-332。
  2. Swetha,C.,Basu,D.,Pachamuthu,K.,Tirumalai,V.,Nair,A.,Prasad,M。和Shivaprasad,P.V。(2018)。 籼稻中与驯化相关的主要表型是由于miRNA介导的漆酶沉默丧失所致。 植物细胞 30(11):2649-2662。
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引用:Swetha, C. and Shivaprasad, P. V. (2019). Extraction and Purification of Laccases from Rice Stems. Bio-protocol 9(7): e3208. DOI: 10.21769/BioProtoc.3208.
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