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Expression and Partial Purification of His-tagged Proteins in a Plant System
植物系统中His标记蛋白的表达和部分纯化

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Abstract

Plant protein expression can be a challenging enterprise in any biochemical or molecular biology research project. Several heterologous systems like bacteria, yeast, insect cells and cell free systems have been used to produce plant proteins for in vitro experiments and structural characterization. However, due to particularities of plant proteins, for example the specific type and abundance of post-translational modifications (e.g. glycosylation), a plant system to express plant proteins is extremely desirable. The use of Nicotiana benthamiana (N. benthamiana) plants for protein expression has proven to be quick and reliable. To illustrate the robustness and rapidity of this system, recent efforts to produce the first protein based drug against the Ebola virus was conducted in N. benthamiana protein expression systems (Choi et al., 2015).
This protocol describes a simple system for the expression and enrichment (affinity purification) of plant apoplastic proteins in N. benthamiana leaves, which was successfully used in the characterization of the Arabidopsis thaliana pectin acetylesterases, PAE8 and PAE9 (de Souza et al., 2014).

Materials and Reagents

  1. Nicotiana benthamiana seeds
  2. Agrobacterium strain GV3101 (obtained from the Lab of Dr. Markus Pauly at UC Berkeley’s Plant and Microbial Biology departmenty)
  3. PRO-MIX® HP MYCORRHIZAE™ soil mix (Promix, catalog number: 20381RG )
  4. Miracle-Gro® Water Soluble All Purpose Plant Food (Scotts)
  5. pART27 expression vector (Gleave, 1992)
  6. Tryptone (MP Biomedicals, catalog number: 1010817 )
  7. Yeast extract (U.S. Biotech Sources, catalog number: Y01PD-500 )
  8. NaCl (Thermo Fisher Scientific, catalog number: S271-3 )
  9. 4'-Hydroxy-3', 5'-dimethoxyacetophenone (acetosyringone) (150 mM in DMSO) (Sigma-Aldrich, catalog number: D134406-1G )
  10. Aluminum foil (Reynolds wrap 76.2 m x 304 mm) (Reynolds Consumer Products Inc.)
  11. Liquid nitrogen
  12. 100x Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific, catalog number: 78429 )
  13. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  14. Bradford reagent (Bio-Rad Protein Assay Dye reagent concentrate) (Bio-Rad Laboratories, AbD Serotec®, catalog number: 500-0006 )
  15. Small columns for Ni-NTA bead wash and elution (any that will fit a 1.5 ml Eppendorf tube, e.g. miniprep column)
  16. 500 µl Vivaspin Column MWCO of 5,000 (Sartorius stedim biotech, catalog number: VS0111 )
  17. Ni-NTA beads (QIAGEN, catalog number: 1018240 )
  18. 96 well plate s for Bradford assay (Thermo Fisher Scientific, catalog number: 80040LE0910 )
  19. 2-(N-morpholino)ethanesulfonic acid (MES) (Sigma-Aldrich, catalog number: M2933 )
  20. MgCl2 (Thermo Fisher Scientific, catalog number: M33-500 )
  21. Sodium phosphate monobasic (Thermo Fisher Scientific, catalog number: S369-500 )
  22. Sodium phosphate dibasic (Thermo Fisher Scientific, catalog number: S374-500 )
  23. Imidazole (Sigma-Aldrich, catalog number: I-2399 )
  24. Lennox LB media (see Recipes)
  25. Infiltration buffer (see Recipes)
  26. Sodium phosphate buffer (see Recipes)
  27. Extraction buffer (see Recipes)
  28. Wash buffer (see Recipes)
  29. Elution buffer (see Recipes)

Equipment

  1. Plant pots (400 ml volume or similar, Gage Durapot) (Merrill's Packaging, catalog number: 03GA-0350S )
  2. Plant growth trays (T.O. Plastics, catalog number: 710245C )
  3. Tall covers that won’t touch the leaves (Acrodome) (Drader Manufactoring Industries Ltd., catalog number: 69973 )
  4. Mortar and pestle
  5. 500 ml culture flasks
  6. Immersion recipient for dipping N. benthamiana plants (e.g. 250 ml beakers)
  7. Metal beads (2.38 mm) (Tool Supply, catalog number: 6230 )
  8. Ball mill (Mixer Mill MM 400 ) (RETSCH, catalog number: MM 400)
  9. Plant growth chambers capable of sustaining 26 °C under long-day conditions (16 h light/8 h dark) with 170-190 µmol m-2 s-1 light intensity.
  10. Spray bottle with water.
  11. Incubator/orbital shaker, capable of 30 °C at 230 rpm incubation for 500 ml culture flaks.
  12. Spectrophotometer capable of OD600 measurements.
  13. Large centrifuge capable of spinning down 250 ml or greater volumes at 5,000 x g for 10 min
  14. Desiccator or vacuum chamber
  15. Vacuum pump (Savant Systems LLC, catalog number: Gel Pump-GP110 )
  16. -80 °C Freezer
  17. Rotating agitator/circular shaker
  18. Table top centrifuges (500-20,800 x g, 4 °C)
  19. Spectrophotometer capable of 96 well plate measurements at 595 nm

Procedure

  1. Plant growth conditions
    The preparation of N. benthamiana plants is a key step in obtaining satisfactory protein expression; plants should be as vigorous as possible to help in their recovery after infiltration and consequent protein production. In this protocol ~6 week old N. benthamiana plants are used for Agrobacterium tumefaciens infiltration.
    1. Grow plants at 26 °C under long-day conditions (16 h light/8 h dark) with 170-190 µmol m-2 s-1 light intensity and optimal humidity of 70%.
    2. Sow seeds in water-soaked soil mix (Promix HP mycorrhizae; 400 ml pots; Video 1) and grow for 2 weeks before transplanting to final destination pots (400 ml).

      Video 1. Detailed description of the procedures for sowing seeds (0 sec), transplanting seedlings (50 sec) and performing the vacuum infiltration (1 min 45 sec)

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    3. Transplant seedlings carefully to preserve as much of the root as possible (Video 1). After transplantation fertilize once with Miracle Grow All-purpose Plant Food (Scotts) according to manufacturer’s recommendation and water as needed. These pots should be grown for another 4 weeks until they are ready for transformation. During the first week of growth young seedlings are kept covered. Multiple fertilization rounds at this stage can cause excessive growth which will produce plants that are too big for infiltration. It can also increase overlap between pots in the trays which will generate problems in the manipulation of the material. Starting at the fifth week it is particularly important to monitor plant development in order to plan for the infiltration procedure. Ideally plants will have 4-5 fully expanded leaves ~7 cm in diameter at 5-6 weeks for infiltration (Video 1).
    4.  When ready for infiltration, if possible, transport plants a few hours in advance to the work site and spray the leaves with abundant water, keeping the plants covered to prevent leaf wilting.

  2. Constructs, Agrobacterium cultures and vacuum infiltration
    The proteins of interest described in this protocol were tagged with 6 histidines at their C- terminus. This construct was cloned into the pART27 binary vector under the control of the 35S promoter (Gleave, 1992). Vectors were transformed into the Agrobacterium strain GV3101 for N. benthamiana transient transformation.
    1. Agrobacterium preparation
      1. Agrobacterium cultures (construct of interest, empty vector and P19) should be started from fresh colonies or glycerol stocks in LB media supplemented with appropriate antibiotics and cultured at 30 °C and 230 rpm. Volumes of at least 200 ml should be used and cultured until reaching OD600 of 1-2 (~ 48 h).
      2. When cultures present increased turbidity, measure OD600 and calculate the necessary dilution so that a final volume of at least 250 ml of OD600 0.7 will be obtained in infiltration buffer for each construct. If necessary allow cultures to grow longer to reach the required amount of cells. When co-infiltrating with P19, a suppressor of gene silencing (Voinnet et al., 2003), the final calculated OD600 of each individual construct should be at least 0.7 (total OD600 will be the sum of both individual ODs). Ideally the final OD600 ratio between construct of interest and P19 should be of 0.7:1.
      3. Spin down cells at 22 °C and 5,000 x g for 10 min.
      4. Discard LB media supernatant by decanting, eliminating as much of the supernatant as possible.
      5. Resuspend cell pellet using infiltration buffer.
      6. Add 500 µl of acetosyringone (4'-Hydroxy-3',5'-dimethoxyacetophenone; 150 mM in DMSO) for every 500 ml of suspended cells in infiltration buffer.
      7. Incubate bacteria for 3-4 h at room temperature before infiltration.
    2. Vacuum infiltration procedure (see Video 1 for details on this section)
      1. Cover the top part of the N. benthamiana pots with aluminum foil. This is to prevent excessive soil loss during the procedure and to keep infiltration buffer as clean as possible, for multiple infiltrations. A square sheet of foil, larger than the plant pot, slit from one of the sides to the center makes an easy to use, disposable cover.
      2. Place the infiltration buffer with cells in a beaker or container that will allow full immersion of the N. benthamiana leaves when dipped.
      3.  Dip the plant aerial part into the cell suspension making sure all the leaves are immersed in the infiltration buffer. Be careful not to break petioles or damage the plant.
      4. Place beaker with plant in a vacuum chamber or desiccator capable of tolerating vacuum pressures. Depending on the size of the container available, multiple plants can be infiltrated simultaneously.
      5. Use a vacuum pump strong enough to produce a vacuum that will pull most of the gas out of the leaves (at least 90 kPa). When vacuum is applied gas bubbles can be observed coming out of the leaves. For every infiltration apply vacuum for 3 min, releasing the vacuum gently, and repeat operation for a total of three times.
      6. Remove the plants from the infiltration buffer. Leaves that were successfully infiltrated should have a translucent or water-soaked appearance. Remove any leaves that were not successfully infiltrated. Leaves that were not fully submerged usually don’t infiltrate very well.
      7. Place plants in a covered tray with plenty of water at the bottom. Return plants to growth chambers.
      8. Remove covers from trays 24-48 h after infiltration, according to how well plants recovered.

  3. Protein extraction and partial purification
    The partial protein purification described here is based on the affinity of the 6x histidine C-terminal tag to nickel-containing resins. The affinity is based on the charges of the two mentioned groups. In plants, this approach has proven to be useful; however it is difficult to obtain pure preparations of the protein of interest using this technique alone. The method described here is able to enrich for the protein of interest but does not produce completely pure fractions. For this reason an “empty vector” protein extraction should be done in parallel to ensure that every downstream experiment using the partially purified protein has a proper negative control.
    1. Protein Extraction
      1. In 3-5 days harvest N. benthamiana leaves for protein extraction by flash-freezing them in liquid nitrogen. Depending on the protein being expressed, shorter incubation periods after infiltration could be attempted.
      2. Pre-grind leaves (empty vector control and construct of interest co-transformed with P19) in a mortar and pestle with liquid nitrogen. This powder can be stored at -80 °C. Pre-grinding can facilitate further processing for protein extraction.
      3. Add 3 metal beads (2.38 mm) to a 2 ml tube.
      4.  Without allowing pre-ground leaves to thaw (work on dry ice and in a cold room if available), place ~ 1 ml of ground material into a 2 ml tube. This volume can be scaled up proportionally if necessary.
      5. Using a bead beater (Retsch ball mill) grind material frozen in liquid nitrogen for 2.5 min, at 25 Hz.
      6.  To 1 ml of extraction buffer add 10 μl of 100x Halt™ Protease Inhibitor Cocktail (0.99x) and 0.14 μl of β-mercaptoethanol (1.98 mM) (final concentrations of 49.5 mM sodium phosphate, 0.99 M NaCl and 9.9 mM imidazole).
      7. Incubate at 4 °C with gentle agitation for 1 h. Use rotating agitator, or similar device that allows the buffer to move in the tube and completely mix the sample.
      8. After one hour remove metal beads with a magnet, always keeping samples on ice.
      9. Pellet plant debris by centrifugation at 4 °C and 20,800 x g for 10 min.
      10. Collect ~ 1.1 ml of supernatant into a new 2 ml tube.
      11. Centrifuge again at 4 °C and 20,800 x g for 10 min, to pellet any carryover leaf debris.
      12. Collect 1 ml of supernatant and transfer to a fresh tube. Depending on the volume being processed it can be a 2 ml tube or larger. Always keep samples on ice.
      13. Measure protein content of the collected supernatant using Bradford assay (Bio-Rad Protein Assay Dye reagent concentrate). It is recommended to use a 96 well plate format with a bovine serum albumin standard curve. The protein measurement here is important to normalize the amount of protein loading onto the affinity beads, 2-3 mg of total protein/ml should be expected. At this stage protein crude extracts can be tested for the presence of the protein of interest using immunoblotting techniques (westerns or dot blots). This is recommended when setting up conditions for protein expression.
    2. Nickel NTA bead preparation
      1. Re-suspend Ni-NTA beads and collect 100 µl into a 1.5 ml tube (50 µl of resin, resin usually compose half the volume of the product).
      2. Spin down at 500 x g for 1 min and remove supernatant.
      3. Wash 3 times with 500 µl extraction buffer, using same centrifugation conditions described above. Final suspension is done in 500 µl extraction buffer.
      4. After washes add 10 µl of resin (110 µl of suspension) to every 1 ml of crude protein supernatant.
    3. Partial protein purification
      1. Incubate for 1 h at 4 °C under gentle agitation to allow tagged proteins to bind to the Ni-NTA beads. During this time undesired precipitation of proteins may occur, in this case an alternative procedure is to bind the tagged proteins to the resin by running the supernatant multiple times through a column containing the beads instead of the batch procedure described.
      2. Spin down to collect beads at 4 °C and 500 x g for 1 min. The beads will form a pellet on the bottom of the tube.
      3. Collect 250 µl of beads and supernatant and place into a small spin column for table top centrifuge with a 2 ml collection tube. Any column that will fit in an Eppendorf-like tube can be used here, since its purpose is just to serve as a support for the Ni-NTA beads. Column material used shouldn’t bind proteins. This step greatly facilitates the procedure and speeds up the washes and elution steps.
      4. Spin down at 4 °C and 500 x g for 1 min.
      5. Wash beads 5 times with 250 µl extraction buffer + protease inhibitors and β-mercaptoethanol (see step C1f). The wash consists of adding the referred volume and discarding the flow through after centrifugation (4 °C and 500 x g for 1 min). Alternatively flow through of the washes can be kept to monitor the presence of the protein of interest using immunoblotting techniques.
      6. Wash 4 times with 200 µl washing buffer
      7. Elute 6 times in 50 µl of elution Buffer into a fresh tube.
      8. Place elution fraction (~ 300 µl) in a 500 µl Vivaspin Column (MWCO of 5,000) for buffer exchange. In this case buffer exchange was necessary due to incompatibility of imidazole and downstream assays, this procedure might not always be necessary.
      9. Spin down at 4 °C and 20,800 x g for 5 min and add 300 µl of 50 mM ammonium formate (pH 4.5) to ~100 µl of sample (4 times dilution, each time). Repeat the procedure for a total of 4 times resulting in the recovery of 200 µl of material containing less than 1 mM imidazole. The protein concentration yield is approximately 0.025 mg/ml for every 1 ml of plant tissue starting material.

Recipes

  1. Lennox LB media
    Dissolve in 1 L water, 10 g of tryptone, 5 g yeast extract and 5 g NaCl
  2. Infiltration buffer
    10 mM MES
    10 mM MgCl2
    pH 5.6
  3. Sodium phosphate buffer (pH 8)
    93.2 ml of 1 M sodium phosphate dibasic
    6.8 ml of 1 M sodium phosphate monobasic
    Add double distilled water to 1 L
  4. Extraction buffer
    1 M NaCl
    50 mM sodium phosphate (pH 8)
    10 mM imidazole
  5. Wash buffer
    300 mM NaCl
    50 mM sodium phosphate (pH 8)
    20 mM imidazole
  6. Elution buffer
    300 mM NaCl
    50 mM sodium phosphate (pH 8)
    150 mM imidazole

Acknowledgements

This protocol is an expansion of that described in de Souza et al. (2014). I would like to thank Marta L. Bjornson for aiding in the revision of the manuscript and Dr. Katayoon Dehesh for laboratory logistical support in the revision process. This work was supported by the Energy Biosciences Institute at UC Berkeley.

References

  1. Choi, W. Y., Hong, K. J., Hong, J. E. and Lee, W. J. (2015). Progress of vaccine and drug development for Ebola preparedness. Clin Exp Vaccine Res 4(1): 11-16.
  2. de Souza, A., Hull, P. A., Gille, S. and Pauly, M. (2014). Identification and functional characterization of the distinct plant pectin esterases PAE8 and PAE9 and their deletion mutants. Planta 240(5): 1123-1138.
  3. Gleave, A. P. (1992). A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol 20(6): 1203-1207.
  4. Voinnet, O., Rivas, S., Mestre, P. and Baulcombe, D. (2003). An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33(5): 949-956.

简介

植物蛋白表达可以是任何生物化学或分子生物学研究项目中的挑战性企业。几种异源系统如细菌,酵母,昆虫细胞和无细胞系统已被用于生产植物蛋白用于体外实验和结构表征。然而,由于植物蛋白的特殊性,例如翻译后修饰(例如糖基化)的特异性类型和丰度,表达植物蛋白的植物系统是非常需要的。已经证明使用本氏烟草(本生烟草)植物进行蛋白质表达是快速和可靠的。为了说明该系统的稳健性和快速性,最近在N中进行了生产针对埃博拉病毒的第一种基于蛋白质的药物的努力。本草案蛋白质表达系统(Choi等人,2015)。
该方案描述了一种简单的系统,用于表达和富集(亲和纯化) > N。本生烟草叶,其被成功地用于拟南芥果胶乙酰酯酶,PAE8和PAE9的表征中(de Souza等人,2014)。

材料和试剂

  1. 本尼特烟草种子
  2. 农杆菌菌株GV3101(从University of UCBekeley's Plant and Microbial Biology department的Markus Pauly博士的实验室获得)
  3. PRO-MIX HP MYCORRHIZAE™土壤混合物(Promix,目录号:20381RG)
  4. Miracle-Gro ®水溶性多用途植物食品(Scotts)
  5. pART27表达载体(Gleave,1992)
  6. 胰蛋白胨(MP Biomedicals,目录号:1010817)
  7. 酵母提取物(U.S.Biotech Sources,目录号:Y01PD-500)
  8. NaCl(Thermo Fisher Scientific,目录号:S271-3)
  9. 4'-羟基-3',5'-二甲氧基苯乙酮(乙酰丁香酮)(150mM,在DMSO中)(Sigma-Aldrich,目录号:D134406-1G)
  10. 铝箔(Reynolds包装76.2m×304mm)(Reynolds Consumer Products Inc.)
  11. 液氮
  12. 100x Halt TM蛋白酶抑制剂混合物(Thermo Fisher Scientific,目录号:78429)
  13. β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  14. Bradford试剂(Bio-Rad Protein Assay Dye reagent Concentrate)(Bio-Rad Laboratories,AbD Serotec ,目录号:500-0006)
  15. 用于Ni-NTA珠粒洗涤和洗脱的小柱(任何适合1.5ml Eppendorf管,例如小量制备柱)的柱子
  16. 500μlVivaspin Column MWCO 5000(Sartorius stedim biotech,目录号:VS0111)
  17. Ni-NTA珠(QIAGEN,目录号:1018240)
  18. 用于Bradford测定的96孔板(Thermo Fisher Scientific,目录号:80040LE0910)
  19. 2-(N-吗啉代)乙磺酸(MES)(Sigma-Aldrich,目录号:M2933)
  20. MgCl 2(Thermo Fisher Scientific,目录号:M33-500)
  21. 磷酸二氢钠(Thermo Fisher Scientific,目录号:S369-500)
  22. 磷酸氢二钠(Thermo Fisher Scientific,目录号:S374-500)
  23. 咪唑(Sigma-Aldrich,目录号:I-2399)
  24. Lennox LB媒体(见配方)
  25. 渗透缓冲液(参见配方)
  26. 磷酸钠缓冲液(见配方)
  27. 提取缓冲液(参见配方)
  28. 洗涤缓冲液(见配方)
  29. 洗脱缓冲液(见配方)

设备

  1. 植物盆(400ml体积或类似物,Gage Durapot)(Merrill's Packaging,目录号:03GA-0350S)
  2. 植物生长盘(T.O.Plastics,目录号:710245C)
  3. 不会接触叶的高盖(Acrodome)(Drader Manufactoring Industries Ltd.,目录号:69973)
  4. 砂浆和杵
  5. 500 ml培养瓶
  6. 浸渍容器的浸渍容器。 本生植物(例如 250ml烧杯)
  7. 金属珠(2.38mm)(工具供应,目录号:6230)
  8. 球磨机(混合机Mill 400)(RETSCH,目录号:MM 400)
  9. 能够在长日照条件(16小时光照/8小时黑暗)下持续26℃的植物生长室,所述条件具有170-190μmolm -1 s -1的光强度 。
  10. 用水冲洗瓶子。
  11. 孵育器/轨道摇床,能够在30℃下以230rpm孵育500ml培养物
  12. 能够测量OD <600>的分光光度计
  13. 大型离心机能够在5,000×g /分钟下旋转250ml或更大体积,持续10分钟
  14. 干燥器或真空室
  15. 真空泵(Savant Systems LLC,目录号:Gel Pump-GP110)
  16. -80°C冰箱
  17. 旋转搅拌器/圆形振动器
  18. 台式离心机(500-20,800×g,4℃)
  19. 分光光度计能够在595nm处进行96孔板测量

程序

  1. 植物生长条件
    N的制备。 本生烟草植物是获得令人满意的蛋白质表达的关键步骤; 植物应该尽可能有力地帮助其在浸润后的恢复和随后的蛋白质生产。 在该协议〜6周龄。 benthamian 植物用于土壤根瘤农杆菌渗入。
    1. 在长日照条件下(16小时光照/8小时黑暗)在26℃下生长植物, 具有170-190μmolm -1 -2光子强度和70%的最佳湿度。< br />
    2. 在水浸泡的土壤混合物(Promix HP mycorrhizae; 400ml盆;   视频1)并生长2周,然后移植到最终目的地   锅(400ml)
      <播种种子(0秒),移植苗(50秒)和进行真空渗入(1分45秒)的程序的详细描述
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    3. 移植幼苗仔细保留尽可能多的根 可能(视频1)。 移植后用奇迹施肥一次 根据制造商生长多用途植物食品(Scotts) 推荐和水。 这些花盆应该种植 另外4周,直到他们准备好转型。 在此期间 第一周生长幼苗被保持覆盖。 多 在这个阶段受精轮次可能导致过度增长   产生对于渗透而言太大的植物。 它也可以增加 在托盘之间的重叠会产生问题 操纵材料。 从第五周开始 特别重要的是监测植物发育以便计划   渗透程序。 理想情况下植物将完全膨胀4-5   叶直径7厘米,5-6周浸润(视频1)
    4.  当准备好渗透时,如果可能,运输植物几个小时 提前到工作现场,用大量的水喷洒叶子, 保持植物覆盖以防止叶枯萎。

  2. 构建体,土壤杆菌培养物和真空浸润 在该方案中描述的感兴趣的蛋白质在其C末端用6个组氨酸标记。将该构建体克隆到在35S启动子控制下的pART27二元载体中(Gleave,1992)。将载体转化到土壤杆菌菌株GV3101中用于N。本氏体瞬时转化。
    1. 农杆菌准备
      1. 土壤杆菌培养物  兴趣,空载体和P19)应从新鲜的殖民地开始 或在补充有合适的抗生素的LB培养基中的甘油储备液  并在30℃和230rpm下培养。至少200毫升的体积应该  使用并培养直至达到1-2(〜48小时)的OD 600
      2. 什么时候 培养物呈现增加的浊度,测量OD 600并计算 使得对于每种构建体在浸润缓冲液中将获得至少250ml OD 600的最终体积0.7 0.7。如果 必要允许培养物生长更长以达到所需量 细胞。当与P19,一种基因沉默抑制基因共浸润 (Voinnet等人,2003),每个个体的最终计算的OD 600 构建体应当至少为0.7(总OD 600将是两者的总和 个体OD)。 理想地,构建体之间的最终OD <600>比率 利息和P19应为0.7:1
      3. 在22℃和5,000xg下旋转电池10分钟。
      4. 通过倾析弃去LB培养基上清液,尽可能多地除去上清液
      5. 使用浸润缓冲液重悬细胞沉淀
      6. 加入500微升乙酰丁香酮 (4'-羟基-3',5'-二甲氧基苯乙酮; 150mM在DMSO中) ml的浸润缓冲液中的悬浮细胞
      7. 在室温下孵育细菌3-4小时,然后浸润。
    2. 真空渗透程序(有关此部分的详情,请参阅视频1)
      1. 覆盖 N的顶部。 本氏烟壶带铝箔。 这是为了防止过程中的过多土壤损失和保持 渗透缓冲液尽可能干净,用于多次渗透。 一个 方片的箔,大于花盆,从一个切开 侧面到中心,使一个易于使用的一次性封面
      2. 地点   具有在烧杯或容器中的细胞的浸润缓冲液 允许完全浸没 N。 benthamiana 在浸泡时离开。
      3.  将植物地上部分浸入细胞悬浮液,确保所有的 叶浸没在浸润缓冲液中。 小心不要打破 叶柄或损坏植物
      4. 与植物在真空中放置烧杯   室或干燥器能够承受真空压力。 根据   对容器的大小可用,多个工厂可以 同时渗透
      5. 使用足够强的真空泵 产生真空,其将大部分气体从叶中抽出(at 至少90kPa)。 当施加真空时,可观察到气泡   出叶。 对于每次渗透施加真空3分钟, 轻轻释放真空,重复操作共三次 次。
      6. 从浸润缓冲液中除去植物。 树叶 成功渗透应该有半透明或 水浸的外观。 删除任何不成功的树叶 渗透。 没有完全淹没的叶子通常不会 渗透非常好。
      7. 将植物放在有盖的托盘中,底部有大量的水。 将植物返回生长室。
      8. 根据植物恢复的情况,在浸润后24-48小时从托盘上取下盖子

  3. 蛋白质提取和部分纯化
    这里描述的部分蛋白质纯化基于6x组氨酸C-末端标签对含镍树脂的亲和力。亲和力基于上述两个基团的电荷。在植物中,这种方法已被证明是有用的;然而,仅使用这种技术难以获得目标蛋白质的纯制剂。本文所述的方法能够富集目标蛋白质,但不产生完全纯的级分。为此,应该平行进行"空载体"蛋白质提取,以确保使用部分纯化的蛋白质的每个下游实验具有适当的阴性对照。
    1. 蛋白质提取
      1. 在3-5天收获。本生植物叶 通过在液氮中快速冷冻蛋白质来提取蛋白质。 根据被表达的蛋白质,更短的孵育期 可以尝试渗透。
      2. 预研磨叶(空 载体对照和用P19共转化的感兴趣的构建体) 砂浆和杵与液氮。这种粉末可以储存在-80℃ C。 预研磨可以促进蛋白质的进一步加工 提取
      3. 向2ml管中加入3个金属珠(2.38mm)
      4.  不允许预地叶解冻(在干冰上工作, 冷室,如果可用),将〜1ml的研磨材料放入2ml 管。 如果需要,可以按比例按比例放大此卷。
      5. 使用珠磨机(Retsch球磨机)在25Hz下在液氮中冷冻2.5分钟来研磨材料。
      6.  向1 ml提取缓冲液中加入10μl100x Halt™蛋白酶 抑制剂混合物(0.99x)和0.14μlβ-巯基乙醇(1.98mM) (终浓度为49.5mM磷酸钠,0.99M NaCl和9.9mM) mM咪唑)
      7. 在4℃下温和搅拌孵育1小时。 使用旋转搅拌器或允许缓冲液移动的类似装置 在试管中并完全混合样品
      8. 一小时后,用磁铁除去金属珠,始终将样品保持在冰上
      9. 通过在4℃和20,800×g离心10分钟来沉淀植物碎片
      10. 收集约1.1毫升上清液到新的2毫升管中
      11. 再次在4℃和20,800×g离心10分钟,以沉淀任何残留叶碎片。
      12. 收集1毫升的上清液,并转移到一个新的管。 根据被处理的体积,它可以是2ml管或更大。   始终将样品保存在冰上。
      13. 测量蛋白质含量 使用Bradford测定法(Bio-Rad Protein Assay Dye 试剂浓缩物)。 建议使用96孔板格式 与牛血清白蛋白标准曲线。 蛋白质测量   对于标准化蛋白质负载量是重要的 亲和珠,应预期2-3mg总蛋白/ml。 在这 可以检测阶段蛋白质粗提物的存在 使用免疫印迹技术(西方或点 印迹)。 这是建立在蛋白质的条件设置 表达。
    2. 镍NTA珠制备
      1. 重新暂停Ni-NTA 珠并收集100μl到1.5ml管(50μl树脂,树脂 通常构成产品体积的一半)。
      2. 在500×g下旋转1分钟,除去上清液
      3. 用500μl提取缓冲液洗涤3次,使用相同的 离心条件。 最终悬浮进行 500μl提取缓冲液
      4. 洗涤后,向每1ml粗蛋白上清液中加入10μl树脂(110μl悬浮液)。
    3. 部分蛋白质纯化
      1. 在4℃下温和搅拌孵育1小时以允许标记 蛋白质结合到Ni-NTA珠。 在此期间不需要 蛋白质的沉淀可能发生,在这种情况下是可选择的 程序是通过运行将标记的蛋白质结合到树脂上 上清液多次通过含有珠子的柱子   的所述批处理程序
      2. 旋转以在4℃和500×g下收集珠子1分钟。 珠子将在管的底部形成颗粒。
      3. 收集250微升的珠子和上清液,并放入一个小旋转 柱用于具有2ml收集管的台式离心机。 任何列 将适合在类似于Eppendorf的管可以在这里使用,因为它 目的只是作为Ni-NTA珠的支撑。 柱 使用的材料不应该结合蛋白质。 这个步骤大大方便了   程序,并加快洗涤和洗脱步骤
      4. 在4℃和500×g下旋转1分钟
      5. 用250μl提取缓冲液+蛋白酶洗涤珠子5次 抑制剂和β-巯基乙醇(参见步骤C1f)。 洗涤包括 添加引用卷并丢弃流过 离心(4℃和500xg,1分钟)。 或者流过 的洗涤液可以保持监测蛋白质的存在 利用免疫印迹技术
      6. 用200μl洗涤缓冲液
        洗涤4次
      7. 在50μl洗脱缓冲液洗脱6次到新管中
      8. 将洗脱级分(〜300μl)置于500μlVivaspin柱(MWCO 为5,000)用于缓冲液交换。 在这种情况下,缓冲液交换 由于咪唑与下游测定的不相容性, 此过程可能不总是必要的。
      9. 在4°C下旋转   和20,800×g 5分钟,并加入300μl50mM甲酸铵(pH值) 4.5)至〜100μl样品(每次稀释4倍)。 重复 程序共4次,导致回收200μl 含有小于1mM咪唑的材料。 蛋白质浓度 每1ml植物组织的产量约为0.025mg/ml 原料。

食谱

  1. Lennox LB媒体
    溶解于1L水,10g胰蛋白胨,5g酵母提取物和5g NaCl中
  2. 渗透缓冲液
    10 mM MES
    10mM MgCl 2/
    pH 5.6
  3. 磷酸钠缓冲液(pH 8)
    93.2ml 1M磷酸氢二钠 6.8ml 1M磷酸二氢钠 加双蒸水至1 L
  4. 提取缓冲区
    1 M NaCl
    50mM磷酸钠(pH8)
    10mM咪唑
  5. 洗涤缓冲液
    300 mM NaCl
    50mM磷酸钠(pH8)
    20mM咪唑
  6. 洗脱缓冲液
    300 mM NaCl
    50mM磷酸钠(pH8)
    150mM咪唑

致谢

该协议是de Souza等人(2014)中描述的扩展。 我要感谢Marta L. Bjornson帮助修订稿件,并感谢Katayoon Dehesh博士在修订过程中提供实验室后勤支持。 这项工作由加州大学伯克利分校的能源生物科学研究所支持。

参考文献

  1. Choi,W.Y.,Hong,K.J.,Hong,J.E.and Lee,W.J。(2015)。 埃博拉疫情的疫苗和药物开发进展 Clin Exp Vaccine Res 4(1):11-16。
  2. de Souza,A.,Hull,P.A.,Gille,S.and Pauly,M。(2014)。 不同植物果胶酯酶PAE8和PAE9及其缺失突变体的鉴定和功能表征。 240(5):1123-1138。
  3. Gleave,A.P。(1992)。 一种具有T-DNA组织结构的多功能双元载体系统,有利于将克隆的DNA有效整合到 植物基因组。 植物分子生物学 20(6):1203-1207。
  4. Voinnet,O.,Rivas,S.,Mestre,P。和Baulcombe,D。(2003)。 基于番茄丛生特技的p19蛋白对基因沉默的抑制的植物中的增强的瞬时表达系统 病毒。植物J 33(5):949-956。
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引用:Souza, A. d. (2015). Expression and Partial Purification of His-tagged Proteins in a Plant System. Bio-protocol 5(17): e1572. DOI: 10.21769/BioProtoc.1572.
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