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Nov 2020

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Apoplastic Expression of CARD1-ecto Domain in Nicotiana benthamiana and Purification from the Apoplastic Fluids
本氏烟草中 CARD1-ecto 结构域的质外体表达及质外体液的纯化   

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Abstract

The protein expression and purification process is an essential initial step for biochemical analysis of a protein of interest. Traditionally, heterologous protein expression systems (such as E. coli, yeast, insect cells, and cell-free) are employed for plant protein expression, although a plant expression system is often desirable for plant proteins, to ensure proper post-translational modifications. Here, we describe a method to express and purify the ectodomain of one of the leucine-rich repeat receptor-like kinase called CARD1/HPCA1, from Nicotiana benthamiana apoplastic fluid. First, we express His-tagged CARD1 ectodomain in the apoplastic space of N. benthamiana by the Agroinfiltration method. Then, we collect apoplastic fluids from the leaves and purify the His-tagged protein by Ni2+-affinity chromatography. In addition to plant-specific post-translational modifications, protein accumulated in the plant apoplastic space, rather than in the cytosolic space, should be kept under an oxidizing environment. Such an environment will help to maintain the property of intrinsic disulfide bonds in the protein of interest. Further, purification from the apoplastic fluids, rather than the total protein extract, will significantly reduce contaminants (for instance RuBisCO) during protein extraction, and simplify downstream processes. We envisage that our system will be useful for expressing various plant proteins, particularly the apoplastic or extracellular regions of membrane proteins.

Keywords: Protein expression and purification (蛋白质表达和纯化), Nicotiana benthamiana (本氏烟草), Leucine-rich repeat receptor-like kinase (富含亮氨酸的重复受体样激酶), Apoplastic fluids (非质体流体), Signal peptide (信号肽)

Background

The use of Nicotiana benthamiana as a host organism for protein expression has increasingly become an attractive system, in addition to well-established expression systems like E. coli, yeast, insect cells, and cell-free. However, it is challenging to obtain near homogeneity protein by one-step affinity purification in the N. benthamiana system, possibly due to the complexity of the total protein extract (Souza, 2015). An alternative option is to ensure that the proteins of interest are expressed and accumulated in specialized compartments, which are spatially separated from contaminants derived from other compartments. Thus, by collecting proteins of interest accumulated in a specific compartment, we can simplify the heterogeneity of the starting material for column chromatography.


Many plant proteins passing through the secretory pathway need to get oxidized and form disulfide bonds, to mature into a stable form (Meyer et al., 2019). The appropriate disulfide bonding is required in many apoplastic proteins or extracellular regions of membrane proteins, including membrane-bound leucine-rich repeat (LRR) receptor-like kinase, and secreted peptide hormones (Meyer et al., 2019). However, the cytoplasm is normally maintained in a reducing environment. It is desirable to accumulate the expressed proteins in the apoplast, and retrieve them with appropriate disulfide bond modifications.


In this protocol, we describe a method to express and purify the ectodomain of an LRR receptor-like kinase CARD1/HPCA1 (CARD1ecto) from N. benthamiana apoplastic fluid. We will primarily focus on its expression and accumulation in the apoplast (Procedure A), its extraction from the apoplastic space (Procedure B), and its purification by Ni2+-affinity chromatography (Procedure C). This system should prove useful not only for CARD1ecto, but also for other plant proteins of interest, particularly the apoplastic or extracellular regions of membrane proteins, which may require redox-related modifications to function correctly.

Materials and Reagents

  1. 1 mL needleless plastic syringe (TERUMO, catalog number: SS-01T)

  2. 20 mL needleless plastic syringe (TERUMO, catalog number: SS-20ESz)

  3. 50 mL centrifuge tube (FALCON, catalog number: 352070)

  4. GD/X syringe filter (PES 0.45 μm) (GE healthcare, catalog number: 6876-2504)

  5. 500 mL Pyrex beaker

  6. 300 mL Pyrex beaker

  7. Vacuum desiccator

  8. 15 mL centrifuge tube (FALCON, catalog number: 352097)

  9. Soil-grown Nicotiana benthamiana

  10. pEAQ-HT plasmid (Sainsbury et al., 2009)

  11. Agrobacterium tumefaciens C58C1 carrying pCH32 (Hamilton et al., 1996; Hellens et al., 2000)

  12. LB Broth (Lennox) (Sigma-Aldrich, catalog number: L7275-500TAB)

  13. Kanamycin (FUJIFILM Wako Chemicals, catalog number: 113-00343)

  14. Rifampicin (FUJIFILM Wako Chemicals, catalog number: 185-01003)

  15. 4'-Hydroxy-3',5'-dimethoxyacetophenone (acetosyringone) (Sigma-Aldrich, catalog number: D134406-25G)

  16. Bis-Tris (Dojindo, catalog number: 6976-37-0)

  17. Tween 20 (polyoxyethylene sorbitan monolaurate) (Nacalai Tesque, catalog number: 35624-15)

  18. cOmpleteTM ULTRA Tablets, EDTA-free, Protease Inhibitor Cocktail (Merck, catalog number: 5892953001)

  19. Sodium Chloride (FUJIFILM Wako Chemicals, catalog number: 195-01663)

  20. Magnesium Chloride (FUJIFILM Wako Chemicals, catalog number: 136-03995)

  21. Immobilized Ni2+-affinity column, HisTrap excel (Cytiva, catalog number: 17371205)

    Note: Whilst any standard Ni2+-affinity column should be acceptable, we recommend using the HisTrap excel column by Cytiva. In this method, the His-tagged protein will be purified in buffer at pH 6.0, given that apoplast space is usually weakly acidic. According to the manufacturer’s instructions, HisTrap excel can capture His-tagged proteins even at pH 6.0.

  22. Superloop, 1/16" fittings (ÄKTAdesign), 50 mL (Cytiva, catalog number: 18111382)

  23. Imidazole (FUJIFILM Wako Chemicals, catalog number: 095-00015)

  24. Vivaspin Turbo 15 (10 kDa molecular weight cut off) (Sartorius, catalog number: VST15T01)

  25. Coomassie protein stain, such as InstantBlue (Expedeon, catalog number: ISB01L)

  26. Agroinfiltration buffer (see Recipes)

  27. Vacuum infiltration buffer (see Recipes)

  28. Equilibration buffer (see Recipes)

  29. Wash buffer (see Recipes)

  30. Elution buffer (see Recipes)

Equipment

  1. Vacuum pump (e.g., ULVAC DTC-41, or equivalent model)

  2. Electroporation system (e.g., Bio-Rad Gene Pulser XCellTM, or equivalent)

  3. Centrifuge with a swing rotor for 50 mL centrifuge tubes (e.g., Hitachi CF16RXII with swing rotor T4SS31, or equivalent)

  4. Centrifuge with a fixed angle rotor for 15 mL centrifuge tubes (e.g., Hitachi CR20GIII with fixed angle rotor R15A, or equivalent)

  5. Fast Protein Liquid Chromatography machine (we used the equivalent of GE healthcare AKTA pure 25 M1 (Cytiva, catalog number 29018227), with optional accessories installed).

    Note: Any fast protein liquid chromatography machine from companies such as Cytiva (https://www.cytivalifesciences.com/) or from Bio-Rad (https://www.bio-rad.com/) are fine.

  6. Spectrophotometer (e.g.,Thermo Fisher Scientific NanodDrop OneC, or equivalent)

  7. Standard SDS-PAGE equipment (such as Mini-PROTEAN® Tetra Cell from Bio-Rad). For the SDS-PAGE protocol, please refer to Green and Sambrook (2014).

Procedure

  1. Expression of His-tagged CARD1 ectodomain in apoplastic space of N. benthamiana

    Note: We expressed non-tagged CARD1ecto, and purified it by conventional column chromatographies in our previous study (Laohavisit et al., 2020). Although it is possible to purify non-tagged proteins, the purification procedure is simpler in His-tagged proteins.

    1. Clone the nucleotide sequence encoding CARD1 from Arabidopsis genomic DNA (amino acids 1–546) into the pEAQ-HT vector, in frame with a His tag at C-terminus (CARD1ecto-His), using standard molecular biology techniques (Figure 1).

      Notes:

      1. The native signal peptide of CARD1 should be intact, so that CARD1ecto-His is secreted into the apoplastic space.

      2. In the pEAQ-HT vector system, a gene of interest is inserted between a modified 5’-untranslated region (UTR) and the 3’-UTR from Cowpea mosaic virus RNA-2, and co-expressed together with silencing suppressor p19 (Sainsbury et al., 2009; Sainsbury and Lomonossoff, 2008). This permits an extremely high-level and rapid production of proteins of interest (see Figure 1).

      3. We used genomic DNA to clone the construct, but cDNA should also work.

      4. For His-tag at C-terminus, pEAQ-HT should be digested with AgeI and SmaI restriction enzymes (Figure 1, see Sainsbury et al., 2009 for details).



      Figure 1. The pEAQ-HT vector map.

      Top, a simplified pEAQ-HT vector map. Bottom, a close-up of the restriction enzyme map.


    2. After sequence confirmation, transform electrocompetent cells of Agrobacterium tumefaciens C58C1 with pEAQ-CARD1ecto-His, using an electroporation system (machine default settings for Agrobacterium tumefaciens are capacitor = 25 μF, pulse controller = 200 Ω, and Voltage = 2.4 kV). Plate out the transformed cells onto LB agar plates supplemented with 100 μg/mL rifampicin and 50 μg/mL kanamycin.

    3. Confirm the Agrobacterium colonies with correct transformation using PCR, and make glycerol stocks for long term storage.

    4. Pick a fresh colony or take a small portion of the glycerol stocks of Agrobacterium into 5 mL of LB liquid media supplemented with 100 μg/mL rifampicin and 50 μg/mL kanamycin and incubate it with shaking at 120 rpm at 28°C overnight.

    5. Dilute 2 mL of overnight culture with 18 mL of fresh LB liquid media supplemented with 100 μg/mL rifampicin and 50 μg/mL kanamycin, and incubate with shaking at 120 rpm and 28°C for 4 h.

    6. Centrifuge the Agrobacterium at 3,000 × g for 5 min, and discard the supernatant.

    7. Add 10 mL of Agroinfiltration buffer, and resuspend the pellet.

    8. Repeat steps 6–7.

    9. Measure OD600 of the suspension and adjust it to 0.3.

    10. Infiltrate the Agrobacterium suspension into whole leaves of 5-weeks-old N. benthamiana from the abaxial side, using 1 mL needleless syringes. The infiltrated area should turn darker in color compared to the unfiltrated area (Yin et al., 2017).

      Note: In our experiment, we infiltrated approximately 30 leaves to obtain approximately 1 mg of highly purified CARD1ecto-His protein. For other proteins of interest, it is recommended that the experimenter perform small-scale pilot experiments (for instance, 8–10 leaves per construct) to ascertain how much protein can be obtained, since different proteins will show different expression levels.


  2. Extraction of apoplastic fluid from N. benthamiana leaves

    1. After 4–7 days post inoculation, harvest the infiltrated N. benthamiana leaves and pile them together, such that the abaxial side of the leaves points upward.

    2. Carefully place the pile of leaves into a 500-mL Pyrex beaker containing 150 mL of vacuum-infiltration buffer. Afterward, place a 300-mL Pyrex beaker on top of the pile, such that leave samples are submerged in buffer (Figure 2).



      Figure 2. Set-up for vacuum infiltration.


    3. Place the assembled beaker (with leave samples) into a vacuum desiccator, and apply a pressure of 60 hPa for 10 min using a pump (or other appropriate equipment). Bubbles should be released from the leaves.

    4. Slowly release the pressure inside the desiccator. We usually allow at least 10 min for the pressure to return to normal.

      Note: In this step, expanded air bubbles in the apoplastic space shrink significantly and vacuum-infiltration buffer will replace this space. The color of the leaves should turn darker as the apoplastic space has been filled with buffer. If you do not notice any differences, this suggests that the vacuum infiltration process has not been successful, and step 3 should be carefully repeated.

    5. Carefully take out individual leaves from the beaker and gently remove excess buffer using paper towels. Set these aside.

    6. Meanwhile, assemble the apoplastic fluid collection unit. This consists of the following materials:

      1. 20-mL single-use needleless plastic syringe with the plunger removed (i.e., only the barrel part is needed)

      2. 50-mL centrifuge tubes, non-skirted

      To assemble the apoplastic fluid collection unit, simply place the barrel part of a 20-mL sized syringe into a 50-mL centrifuge tube (Figure 3A).



      Figure 3. Set-up of the apoplastic fluid collection unit.


    7. Per apoplastic fluid collection unit, take 6–8 leaves from step 5 and separate them into two piles (3–4 leaves per pile). Next, carefully roll the leaves from one pile (from the base to the apex of the leaf) and place this into the barrel part of the apoplastic fluid collection unit. The abaxial or adaxial sides can be faced in any direction. Repeat for the second pile (Figure 3B).

      Note: For our experiment, we used approximately 30 leaves to purify CARD1ecto-His protein, which corresponded to 6 apoplastic fluid collection units.

    8. Place the apoplastic fluid collection units (with leaves) in a swing rotor, and centrifuge at 1,500 × g and 4°C for 10 min.

      Note: Centrifugation should be performed using a swing rotor, to maximize recovery of the apoplast fluid.

    9. Apoplastic fluid should appear at the bottom of the 50-mL centrifuged tubes. Transfer the fluid to a separate tube and centrifuge again at 1,500 × g and 4°C for 3 min, to ensure all fluid has been extracted. Finally, pool all the apoplastic fluid into a single tube.

    10. Centrifuge the collected apoplastic fluid from step 9 at 10,000 × g and 4°C for 10 min. Collect the supernatant, and centrifuge it again at 10,000 × g and 4°C for 10 min.

    11. Filter the supernatant with a GD/X syringe filter. The resulting filtrate is now ready for the next step of purification.

      Note: The filter unit should have a 0.45 µm pore size.


  3. CARD1ecto-His purification by Ni2+-affinity chromatography

    Note: Monitor the absorbance at 280 nm to detect proteins throughout the purification process.

    1. Connect a HisTrap excel column (capacity of 5 mL) to an FPLC system (such as the AKTA pure), and wash this column with 25 mL of ultrapure water (5 column volumes, CV) at 5 mL/min (1 CV/min).

      Note: Wash volume and the flow rate should be adjusted according to the column size used.

    2. Equilibrate the column with 25 mL (5 CV) of equilibration buffer at 5 mL/min (1 CV/min).

    3. Load the apoplastic fluid sample onto the column with a superloop at 2.5 mL/min (0.5 CV/min).

      Note: Depending on your FPLC machine configuration and your sample volume, you can also load your sample manually.

    4. Wash the column with 25 mL (5 CV) of wash buffer at 5 mL/min (1 CV/min).

    5. Elute the His-tagged protein with 25 mL (5 CV) of elution buffer at 5 mL/min (1 CV/min).

    6. Pool the peak fractions in one sampling tube and check the purity of CARD1ecto-His by SDS-PAGE, followed by Coomassie brilliant blue staining (InstantBlue; Expedeon) (Figure 4).



      Figure 4. SDS-PAGE analysis of purified CARD1ecto-His.

      Lane 1: Apoplastic fluid from intact N. benthamiana leaves. Lane 2: Apoplastic fluids from the CARD1ecto-His expressing N. benthamiana leaves. Lane 3: Purified CARD1ecto-His by Ni2+-affinity chromatography. The gel was stained with Coomassie brilliant blue (InstantBlue).


    7. Measure protein concentration by absorbance at 280 nm, using a spectrophotometer (such as NanoDrop OneC).

    8. (Optional) Apply the pooled fractions in an ultrafiltration unit with a 10-kDa cut-off membrane (Vivaspin turbo 15) for buffer exchange and/or concentration.

      Note: When buffer exchanging or sample concentration are necessary for downstream processes.

Recipes

  1. Agroinfiltration buffer

    10 mM MES/NaOH (pH 5.6)

    10 mM MgCl2

    150 μM acetosyringone

  2. Vacuum infiltration buffer

    20 mM Bis-Tris/HCl (pH 6.0)

    0.01% Tween20

    1× cOmpleteTM Protease Inhibitor Cocktail

  3. Equilibration buffer

    20 mM Bis-Tris/HCl (pH 6.0)

    300 mM NaCl

  4. Wash buffer

    20 mM Bis-Tris/HCl (pH 6.0)

    300 mM NaCl

    5 mM Imidazole

  5. Elution buffer

    20 mM Bis-Tris/HCl (pH 6.0)

    300 mM NaCl

    300 mM Imidazole

Acknowledgments

This protocol was adapted from our published work (Laohavisit et al., 2020). This work was supported by JSPS Grant-in-Aid for 17H06172 and 20H05909 to K.S.

Competing interests

The authors declare no competing interests.

References

  1. Green, M.R. and Sambrook, J. (2014). Molecular Cloning: A Laboratory Manual (4th Edition). Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.
  2. Hamilton, C.M., Frary, A., Lewis, C. and Tanksley, S.D. (1996). Stable transfer of intact high molecular weight DNA into plant chromosomes. Proc Natl Acad Sci U S A 93(18): 9975-9979.
  3. Hellens, R., Mullineaux, P. and Klee, H. (2000). Technical Focus: a guide to Agrobacterium binary Ti vectors. Trends Plant Science 5(10): 446-451.
  4. Laohavisit, A., Wakatake, T., Ishihama, N., Mulvey, H., Takizawa, K., Suzuki, T. and Shirasu, K. (2020). Quinone perception in plants via leucine-rich-repeat receptor-like kinases. Nature 587(7832): 92-97.
  5. Meyer, A.J., Riemer, J. and Rouhier, N. (2019). Oxidative protein folding: state-of-the-art and current avenues of research in plants. New Phytol 221(3): 1230-1246.
  6. Sainsbury, F. and Lomonossoff, G. P. (2008). Extremely high-level and rapid transient protein production in plants without the use of viral replication. Plant Physiol 148(3): 1212-1218.
  7. Sainsbury, F., Thuenemann, E. C. and Lomonossoff, G. P. (2009). pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J 7(7): 682-693.
  8. Souza, A. D. (2015). Expression and partial purification of His-tagged proteins in a plant system. Bio-protocol 5(17). e1572.
  9. Yin, K., Han, T. and Liu, Y. (2017). Use of Geminivirus for delivery of CRISPR/Cas9 components to tobacco by Agro-infiltration. Bio-protocol 7(7): e2209.

简介

【摘要】蛋白质的表达和纯化过程是感兴趣的蛋白质生化分析的必要的初始步骤。 传统上,植物蛋白表达采用外源蛋白表达系统(如大肠杆菌、酵母、昆虫细胞和无细胞),尽管植物蛋白通常需要植物表达系统,以确保正确的翻译后修饰。 在这里,我们描述了一种方法,表达和纯化一个富含亮氨酸重复受体样激酶CARD1/HPCA1的外域,从本氏烟的离体液。 首先,我们通过Agroinfiltration的方法在本氏氮芥的胞外空间表达带有his标记的CARD1胞外结构域。 然后,我们从叶片中收集外质体液,通过Ni2+亲和层析纯化his标记的蛋白。 除了植物特异的翻译后修饰外,在植物外质空间而不是在细胞质空间中积累的蛋白质应该保持在氧化环境下。 这样的环境将有助于维持感兴趣蛋白质内在二硫键的性质。 此外,从离体液体中纯化,而不是从总蛋白提取物中纯化,将显著减少蛋白质提取过程中的污染物(例如RuBisCO),并简化下游过程。 我们设想我们的系统将用于表达各种植物蛋白,特别是膜蛋白的胞外或胞外区域。

背景

大肠杆菌、酵母、昆虫细胞和无细胞等成熟的表达系统外,使用本氏烟草作为蛋白质表达的宿主生物已越来越成为一种有吸引力的系统。然而,在本氏烟草系统中通过一步亲和纯化获得接近同质的蛋白质具有挑战性,这可能是由于总蛋白质提取物的复杂性(Souza, 2015) 。另一种选择是确保感兴趣的蛋白质在专门的隔间中表达和积累,这些隔间在空间上与来自其他隔间的污染物分开。因此,通过收集在特定隔室中积累的感兴趣的蛋白质,我们可以简化柱层析起始材料的异质性。
许多通过分泌途径的植物蛋白需要被氧化并形成二硫键,才能成熟为稳定的形式(Meyer et al. , 2019) 。许多质外体蛋白或膜蛋白的细胞外区域需要适当的二硫键,包括膜结合的 富含亮氨酸重复 (LRR) 受体样激酶和分泌肽激素(Meyer et al. , 2019) 。然而,细胞质通常维持在还原环境中。需要在质外体中积累表达的蛋白质,并通过适当的二硫键修饰来回收它们。
在本协议中,我们描述了一种表达和纯化LRR 受体样激酶 CARD1/HPCA1(CARD1ecto)的胞外域的方法。 质外体液。我们将主要关注其在质外体中的表达和积累(程序A),其从质外体空间的提取(程序B),以及通过Ni 2+ -亲和色谱法对其进行纯化(程序C)。该系统应该证明不仅对 CARD1ecto 有用,而且对其他感兴趣的植物蛋白也有用,特别是膜蛋白的质外体或细胞外区域,这可能需要与氧化还原相关的修饰才能正常工作。

关键字:蛋白质表达和纯化, 本氏烟草, 富含亮氨酸的重复受体样激酶, 非质体流体, 信号肽



材料和试剂


1. 1 mL无针塑料注射器(TERUMO,目录号:SS-01T)
2. 20 mL无针塑料注射器(TERUMO,目录号:SS-20ESz)
3. 50 mL离心管(FALCON,目录号:352070)
4. GD/X 注射器过滤器(PES 0.45 μm )(GE Healthcare,目录号: 6876-2504)
5. 500 毫升耐热玻璃烧杯
6. 300 毫升耐热玻璃烧杯
7. 真空干燥器
8. 15 mL离心管(FALCON,目录号:352097)
9. 土生烟草_
10. pEAQ -HT 质粒 (Sainsbury et al. , 2009)
11. 携带 pCH32 的根癌农杆菌C58C1 (Hamilton et al. , 1996; Hellens 等。 , 2000)
12. LB Broth(Lennox)(Sigma-Aldrich,目录号:L7275-500TAB)
13. 卡那霉素(FUJIFILM Wako Chemicals,目录号:113-00343)
14. 利福平(FUJIFILM Wako Chemicals,目录号:185-01003)
15. 4'-羟基-3',5'-二甲氧基苯乙酮(乙酰丁香酮) (Sigma-Aldrich,目录号: D134406-25G)
16. Bis-Tris( Dojindo ,目录号:6976-37-0)
17. 吐温20(聚氧乙烯 山梨糖醇单月桂酸酯)( Nacalai Tesque ,目录号:35624-15 )
18. cOmplete TM ULTRA 片剂,不含 EDTA,蛋白酶抑制剂混合物(Merck,目录号:5892953001)
19. 氯化钠(FUJIFILM Wako Chemicals,目录号: 195-01663)
20. 氯化镁(FUJIFILM Wako Chemicals,目录号: 136-03995)
21. 固定化Ni 2+ -亲和柱, HisTrap excel( Cytiva ,目录号:17371205 )


 
22. Superloop ,1/16" 接头( ÄKTAdesign ),50 mL( Cytiva ,目录号:18111382)
23. 咪唑(FUJIFILM Wako Chemicals,目录号:095-00015)
24. Vivaspin Turbo 15(10 kDa 截留分子量)(Sartorius,目录号:VST15T01)
25. 考马斯亮蓝蛋白染色剂,例如InstantBlue ( Expedeon ,目录号: ISB01L )
26. 农杆菌浸润缓冲液(见配方)
27. 真空浸润缓冲液(见配方)
28. 平衡缓冲液(见配方)
29. 洗涤缓冲液(见配方)
30. 洗脱缓冲液(参见配方)




设备


1. 真空泵(例如ULVAC DTC-41 或同等型号)
2. 电穿孔系统(例如Bio-Rad Gene Pulser XCell TM或同等产品)
3. 带有用于 50 mL离心管的摆动转子的离心机(例如,带有摆动转子 T4SS31 的 Hitachi CF16RXII,或同等产品)
4. 用于 15 mL 离心管的带有固定角转子的离心机(例如,带有固定角转子 R15A 的 Hitachi CR20GIII,或同等产品)
5. 快速蛋白质液相色谱仪(我们使用相当于 GE Healthcare AKTA pure 25 M1( Cytiva ,目录号 29018227),安装了可选附件)。
注意:任何来自Cytiva ( https://www.cytivalifesciences.com/ ) 或 Bio-Rad ( https://www.bio-rad.com/ ) 等公司的快速蛋白质液相色谱仪都可以。
6. 分光光度计(例如Thermo Fisher Scientific NanodDrop 一个C或同等学历)
7. 标准 SDS-PAGE 设备(例如 Bio-Rad 的 Mini-PROTEAN ® Tetra Cell)。有关 SDS-PAGE 协议,请参阅 Green 和 Sambrook (2014)。




程序


A. His-tagged CARD1 胞外域在本氏烟草质外体空间的表达


 


1. 编码 CARD1 的核苷酸序列从拟南芥基因组 DNA(氨基酸 1-546)克隆到pEAQ -HT 载体中,在 C 末端带有 His 标签(CARD1ecto-His)的框架中(图 1)。


 




 


图 1. pEAQ -HT 矢量图。 
顶部,简化的pEAQ -HT 矢量图。底部,限制酶图的特写。


2. 序列确认后,使用电穿孔系统用 pEAQ-CARD1ecto-His转化农杆菌C58C1 的电感受态细胞(农杆菌的机器默认设置为电容器 = 25 μF ,脉冲控制器 = 200 Ω,电压 = 2.4 kV)。将转化细胞铺板到 LB 琼脂板上,辅以 100 μg /mL 利福平和50 μg / mL 卡那霉素。
3. PCR确认农杆菌菌落的正确转化,并制作甘油储备以供长期储存。
4. 挑选一个新鲜的菌落或取一小部分农杆菌甘油原液到5 mL 的 LB 液体培养基中,辅以 100 μg /mL 利福平和 50 μg /mL 卡那霉素,并在 28°C 下以 120 rpm 摇动孵育过夜。
5. 补充有 100 μg /mL 利福平和 50 μg /mL卡那霉素的新鲜 LB液体培养基稀释 2 mL 过夜培养物,并在 120 rpm 和 28°C 下振荡孵育 4 小时。
6. 农杆菌以 3,000 × g离心5分钟,弃去上清液。
7. 添加 10 mL 的 Agroinfiltration缓冲液,并重新悬浮颗粒。
8. 重复步骤 6-7。
9. 测量悬浮液的 OD 600并将其调整为 0.3。
10. 农杆菌悬浮液从背面渗透到 5 周大的N.benthamiana的整个叶子中。与未过滤区域相比,浸润区域的颜色应该变深( Yin等人,2017)。 


 


B. 氏烟草叶中提取质外体液


1. 后4-7 天后,收获渗透的本氏烟草叶子并将它们堆放在一起,使叶子的背面朝上。
2. 小心地将叶子堆放入含有 150 mL 真空渗透缓冲液的 500 mL Pyrex 烧杯中。之后,将 300 毫升 Pyrex 烧杯放在堆的顶部,使留下的样品浸没在缓冲液中(图 2)。




 


图 2. 真空渗透设置。


3. 将组装好的烧杯(带有留样)放入真空干燥器中,并使用泵(或其他适当的设备)施加 60 hPa的压力 10 分钟。气泡应该从叶子中释放出来。
4. 慢慢释放干燥器内的压力。我们通常允许至少 10 分钟让压力恢复正常。


 


5. 小心地从烧杯中取出单个叶子,并使用纸巾轻轻去除多余的缓冲液。把这些放在一边。
6. 同时,组装非质体流体收集单元。这包括以下材料:
• 20-mL 一次性无针塑料注射器,柱塞已移除(即,仅需要针筒部分)
• 50-mL 离心管,无裙边
要组装非质体流体收集装置,只需将 20 mL 大小的注射器的筒部分放入 50 mL 离心管中(图 3A)。




 


图 3.质外体流体收集单元的设置。


7. 每个质外体流体收集单元,从步骤 5 中取出 6-8片叶子,并将它们分成两堆(每堆 3-4 片叶子)。接下来,小心地将叶子从一堆(从底部到叶子的顶点)卷起来,并将其放入非质体流体收集单元的桶部分。背轴或近轴侧可以朝向任何方向。重复第二堆(图 3B)。


 


8. 将质外流体收集单元(带叶子)放入摆动转子中,并在 1,500 × g和 4 °C 下离心10 分钟。


 


9. 质外体液应出现在 50 mL 离心管的底部。将流体转移到单独的管中,并在 1,500 × g和 4 °C 下再次离心3 分钟,以确保已提取所有流体。最后,将所有质外体液汇集到一个管中。
10. 在 10,000 × g和4 °C 下将步骤 9 中收集的非质体流体离心10 分钟。收集上清液,在 10,000 × g和 4 °C 下再次离心10 分钟。
11. 用GD/X 注射器过滤器过滤上清液。所得滤液现已准备好进行下一步纯化。


 


C. 通过 Ni 2+亲和层析纯化 CARD1ecto-His


 


1. 将HisTrap excel 柱(容量为 5 mL)连接到 FPLC 系统(例如 AKTA pure ),并用 25 mL 超纯水(5 柱体积,CV)以 5 mL/min(1 CV/min)清洗该柱)。


 


2. 用 25 mL (5 CV) 的平衡缓冲液以 5 mL/min (1 CV/min)平衡色谱柱。
3. 以 2. 5 mL/min (0.5 CV/min)的速度将非质体流体样品加载到带有superloop的柱上。


 


4. (5 CV) 的洗涤缓冲液以 5 mL/min (1 CV/min)的速度洗涤色谱柱。
5. 用 25 mL (5 CV) 的洗脱缓冲液以5 mL/min (1 CV/min)洗脱 His 标签蛋白。
6. 将峰组分汇集在一个采样管中,通过 SDS-PAGE 检查 CARD1ecto-His 的纯度,然后进行考马斯亮蓝染色( InstantBlue ; Expedeon ) (图 4)。




 


图 4.纯化 CARD1ecto-His 的 SDS-PAGE 分析。 
泳道 1:来自完整的本氏烟草叶子的质外体液。泳道 2:来自CARD1ecto -His 表达N.benthamiana叶的质外体液。泳道 3:通过Ni 2+亲和层析纯化的 CARD1ecto-His 。凝胶用考马斯亮蓝 ( InstantBlue ) 染色。


7. 使用分光光度计(例如NanoDrop )在 280 nm 处通过吸光度测量蛋白质浓度 一C )。
8. (可选)在具有 10 kDa 截止膜 ( Vivaspin turbo 15) 的超滤装置中应用汇集的级分,以进行缓冲液交换和/或浓缩。


 




食谱


1. 农用浸润缓冲液
10 mM MES/NaOH (pH 5.6)
10 毫米氯化镁2
150微米 乙酰丁香酮


2. 真空浸润缓冲液
20 mM Bis-Tris/HCl (pH 6.0)
0.01% 吐温20
1 × cOmplete TM蛋白酶抑制剂混合物


3. 平衡缓冲液
20 mM Bis-Tris/HCl (pH 6.0)
300 毫米氯化钠


4. 洗涤缓冲液
20 mM Bis-Tris/HCl (pH 6.0)
300 毫米氯化钠
5 mM 咪唑
5. 洗脱缓冲液
20 mM Bis-Tris/HCl (pH 6.0)
300 毫米氯化钠
300 毫米咪唑




致谢


该协议改编自我们已发表的工作( Laohavisit 等。 , 2020)。这项工作得到了 JSPS Grant-in-Aid 17H06172和 20H05909的支持 到堪萨斯




利益争夺


作者声明没有竞争利益。


参考


Green, MR 和 Sambrook, J. (2014)。分子克隆:实验室手册(第 4版)。纽约冷泉港:冷泉港实验室出版社。
Hamilton, CM, Frary , A., Lewis, C. 和Tanksley , SD (1996)。将完整的高分子量 DNA 稳定转移到植物染色体中。 Proc Natl Acad Sci USA 93(18): 9975-9979。
Hellens , R.、 Mullineaux , P. 和 Klee, H. (2000)。技术重点:农杆菌二元 Ti 载体指南。 趋势植物科学5(10):446-451。
Laohavisit , A.、 Wakatake , T.、 Ishihama , N.、Mulvey, H.、Takizawa, K.、Suzuki, T. 和Shirasu , K. (2020)。通过富含亮氨酸的重复受体样激酶在植物中对醌的感知。 自然587(7832):92-97。
Meyer, AJ, Riemer, J. 和Rouhier , N. (2019)。氧化蛋白质折叠:植物研究的最新和当前途径。 新植物醇221(3):1230-1246。
Sainsbury, F. 和Lomonossoff , GP (2008)。在不使用病毒复制的情况下,在植物中实现极高水平和快速的瞬时蛋白质生产。 植物生理学148(3):1212-1218。
Sainsbury, F., Thuenemann , EC 和Lomonossoff , GP (2009)。 pEAQ:多功能表达载体,用于在植物中轻松快速地瞬时表达异源蛋白。 植物生物技术J 7(7):682-693。
苏萨,公元(2015 年)。 His 标签蛋白在植物系统中的表达和部分纯化。生物协议5(17)。 e1572。
Yin, K.、Han, T. 和 Liu, Y. (2017)。使用双子病毒通过农业渗透将 CRISPR/Cas9 成分递送至烟草。 生物协议7(7):e2209。
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引用:Ishihama, N., Laohavisit, A., Takizawa, K. and Shirasu, K. (2022). Apoplastic Expression of CARD1-ecto Domain in Nicotiana benthamiana and Purification from the Apoplastic Fluids. Bio-protocol 12(8): e4387. DOI: 10.21769/BioProtoc.4387.
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