Monoclonal Antibody Purification (Nicotiana benthamiana Plants)

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Antimicrobial Agents and Chemotherapy
May 2013



Plant-based expression systems provide an alternative biomanufacturing platform for recombinant proteins (Matoba et al., 2011). In particular, plant virus-based vectors can overexpress proteins within days in the leaf tissue of Nicotiana benthamiana (N. benthamiana). To overcome the issues of genetic instability and limited infectivity of recombinant viruses, Agrobacterium-mediated delivery of “deconstructed” virus vectors has become the mainstay for the production of large and/or multicomponent proteins, such as immunoglobulin (Ig)G monoclonal antibodies (mAbs). Here, we describe a method of producing human IgG mAbs in N. benthamiana using the tobamoviral replicon vector magnICON®. The vector can express up to a few hundred mg of a mAb per kg of leaf material in 7 days. A representative case for the broadly neutralizing anti-HIV and anti-influenza mAbs, VRC01 and CR6261 respectively, is shown (Hamorsky et al., 2013). Leaf tissue is homogenized and the extract is clarified by filtration and centrifugation. The mAb is purified by fast protein liquid chromatography (FPLC) using Protein A affinity and Phenyl HP hydrophobic interection resins.

Keywords: Monoclonal antibody (单克隆抗体), Nicotiana benthamiana (烟草), Plant virus vector (植物病毒载体)

Materials and Reagents

  1. Agrobacterium tumefaciens electrocompetent cells (strain GV3101)
  2. MagnICON® plasmid (Icon Genetics GmbH, catalog number: pICH38099 ) (Marillonnet et al., 2004; Giritch et al., 2006)
  3. Rifampicin (Sigma-Aldrich, catalog number: R3501 )
  4. Gentamicin (Sigma-Aldrich, catalog number: G1264 )
  5. Kanamycin (Sigma-Aldrich, catalog number: K1876 )
  6. Yeast Extract (Fisher Scientific, catalog number: BP1422 )
  7. Nutrient Broth (BD, catalog number: 231000 )
  8. UV/Vis cuvette (VWR International, catalog number: 77776-745 )
  9. MES (EMD Millipore, catalog number: 475894 )
  10. Magnesium sulfate (EMD Millipore, catalog number: MX0070-3 )
  11. Sodium phosphate (EMD Millipore, catalog number: SX0710-1 )
  12. Sodium chloride (Sigma-Aldrich, catalog number: S9888 )
  13. Sodium hydroxide
  14. Ascorbic acid (Fisher Scientific, catalog number: BP351 )
  15. 1M Tris buffer (pH 8.0)
  16. Glycine (Fisher Scientific, catalog number: BP381-5 )
  17. L-Arginine (Sigma-Aldrich, catalog number: A5006 )
  18. Dulbecco’s Phosphate Buffered Saline (DPBS) (Gibco®, catalog number: 14190-144 )
  19. LB agar plate (see Recipes)
  20. YenB media (see Recipes)
  21. Infiltration buffer (see Recipes)
  22. Extraction buffer (see Recipes)
  23. Protein A Elution buffer (see Recipes)
  24. Protein A Equilibration/Wash buffer (see Recipes)
  25. Phenyl HP Equilibration/Wash buffer (see Recipes)
  26. Phenyl HP Elution buffer (see Recipes )


  1. Amicon Ultra centrifugal filter, 30 K (EMD Millipore, catalog number: UFC903024 )
  2. Electroporation cuvette (USA Scientific, catalog number: 9104-1050 )
  3. Bottle top filter unit (VWR International, catalog number: 73520-986 )
  4. Beveled flask (VWR International, catalog number: 4446-500 )
  5. Centrifuge bottles (Thermo Fisher Scientific, catalog number: 3141-0250 )
  6. HiTrap Protein A HP column (General Electric Company, catalog number: 17-0403-03 )
  7. Phenyl HP column (General Electric Company, catalog number: 17-5195-01 )
  8. Multiporator (Eppendorf, Bacteria module, catalog number: 4308 805.005 )
  9. 28 °C Incubator (Fisher Scientific)
  10. Benchtop orbital shaker (Thermo Fisher Scientific, catalog number: SHKE4450 )
  11. UV/Vis spectrophotometer (Beckman Coulter, catalog number: DU800 )
  12. Avanti® J-26 XP Centrifuge (Beckman Coulter, catalog number: 393124 )
  13. Infiltration apparatus (Bel-Art Products, catalog number: F420250000 )
  14. Vacuum pump (William H. Welch Medical Library, catalog number: 8890A-75 )
  15. Growth chamber
  16. Blender (Waring Pro, catalog number: 7011HS )
  17. AKTA purifier (General Electric Company, catalog number: 28-4062-66 )


  1. Transform electrocompetent agrobacteria cells with plant-expressing viral vector DNA plasmid containing the sequence for the mAb of interest at 2,000 V for 5 msec.
  2. After shaking transformed cells for 1 h at 28 °C to establish antibiotic resistance, streak cells on LB agar plates (containing 30 μg/ml Rifampicin, 50 μg/ml Gentamicin and 50 μg/ml Kanamycin) and grow in 28 °C incubator for 3 - 4 days.
  3. Pick a single transformed bacterial colony and grow in 5 ml of YenB media plus 30 μg/ml Rifampicin and 50 μg/ml Kanamycin at 28 °C and 225 rpm overnight.
  4. Transfer 5 ml starter culture to large flask containing 95 ml fresh YenB media plus the same antibiotics and grow at 28 °C and 225 rpm overnight.
  5. Measure the optical density at 600 nM using UV/Vis Spectrophotometer using YenB media as a blank. Calculate the volume of the large culture required for a final OD600 equal to 0.03 in the total volume of infiltration buffer needed (1.6 L).
  6. Centrifuge calculated volume of bacterial culture at 5,000 x g, room temperature for 15-30 min. Re-suspend the pelleted bacteria in any volume of infiltration buffer and dilute to final volume of 1.6 L.
    Note: For example, if measure OD600 is 1.25 then the following calculation would be made
    (1.25) x = (0.03) (1.6 L)
    x = 38.4 ml of bacterial culture would need to centrifuged then re-suspended in infiltration buffer.
  7. Transfer the re-suspended bacteria to vacuum apparatus and fill to final volume. Apply 25 inches Hg of pressure to all the leaves of an entire N. benthamiana plant (between 26-30 days old) for 2 min then release the vacuum causing the bacterial inoculum to enter the leaves (Figure 1). The same infiltration buffer can be re-used for up to approximately 16 plants without any noticeable depreciation in inoculation.
    Note: Efforts should be made to keep plant soil from entering infiltration buffer but it will not interfere with successfully infecting plants.

    Figure 1. Vacuum Infiltration. The mAb vector is delivered via A. tumefaciens into N. benthamiana leaves using vacuum infiltration (see above, procedure step 7).

  8. Plants are grown post-infiltration the same as before infiltration, at 27 °C and > 50% humidity following a 16 h daytime/8 h nighttime schedule. Water the plants every other day.
  9. At 7 days post infiltration (dpi), harvest the infected leaves expressing mAb by cutting the leaves at the base of the stem and measure the total mass. Inoculated leaves are distinguishable by the mosaic phenotype displayed after 4-5 days post infiltration compared to non-inoculated leaves.
  10. mAb is extracted in cold extraction buffer by using an industrial blender while mixing 2 ml buffer for every 1 g leaf tissue.
    Note: For the remaining duration of the procedure, all efforts are made to ensure the mAb remains at 4 °C including extraction and purification.
  11. The leaf extract is centrifuged at 15,000 x g for 10 min at 4 °C to pellet plant debris.
  12. The partially clarified extract is pH adjusted to 7.0 using sodium hydroxide and centrifuged at 15,000 x g for 10 min at 4 °C to pellet starches and small plant debris.
  13. The final step of extraction requires the clarified extract to be passed through a 0.2 µm bottle top filter using a vacuum.
  14. mAb is purified initially by using a HiTrap Protein A HP column from GE Healthcare via FPLC on an AKTA purifier. The column is equilibrated with 10 column volume (CV) of Protein A Equilibration/Wash buffer.
  15. mAb clarified extract is loaded at 2 ml/min. After the extract has been loaded to the column, unbound proteins are washed out of the column using Protein A Equilibration/Wash buffer (10 CV).
  16. Protein A Elution buffer is used to elute mAb from the Protein A column using a step gradient to 100% Protein A Elution buffer for 10 CV. The pH is adjusted from 3.0 to 7.0 using 1 M Tris buffer (pH 8.0) for the fractions containing mAb.
    Note: A step gradient is programmed into the method run so that the AKTA purifier transitions from 0% elution buffer to 100% elution buffer in a single step.
  17. Following Protein A purification, a HiTrap Phenyl HP column from GE Healthcare is utilized. The column is equilibrated with 10 CV in Phenyl HP Equilibration/Wash buffer.
  18. Protein A eluted mAb is diluted 1:10 in Phenyl HP Equilibration/Wash buffer and loaded at 2 ml/min. Unbound protein is washed off of the column using 10 CV Phenyl HP Equilibration/Wash buffer.
  19. Phenyl HP Elution buffer is used to elute mAb from the column using a linear gradient from 0% Phenyl HP Elution buffer to 100% Phenyl HP Elution buffer over 30 CV followed by an additional 5 CV at 100% Phenyl HP Elution buffer.
    Note: A linear gradient is programmed into the method run so that the AKTA purifier controls the incremental transition from 0% elution buffer to 100% elution buffer for which the entire length of elution is 30 CV.
  20. Purified mAb is formulated into dulbecco’s phosphate buffered saline (DPBS) pH 7.2. Purity is analyzed by densitometry of an overloaded Coomassie-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions.
  21. Activity is determined by antigen capture enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance and in vitro virus neutralization assays.


  1. LB agar plate
    30 μg/ml Rifampicin
    50 μg/ml Gentamicin
    50 μg/ml Kanamycin
  2. YenB media (for 1 L)
    Yeast Extract 7.5 g
    Nutrient Broth 8.0 g
    pH 7.0
  3. Infiltration buffer
    10 mM MES
    10 mM Magnesium Sulfate
    pH 5.5
  4. Extraction buffer
    100 mM Sodium Phosphate
    100 mM Sodium Chloride
    40 mM Ascorbic Acid
    pH 6.0
  5. Protein A Elution buffer
    100 mM Glycine
    200 mM L-Arginine
    pH 3.0
  6. Protein A Equilibration/Wash buffer
    20 mM Sodium Phosphate
    pH 7.0
  7. Phenyl HP Equilibration/Wash buffer
    50 mM Sodium Phosphate
    1 M Ammonium Sulfat
    pH 7.0
  8. Phenyl HP Elution buffer
    50 mM Sodium Phosphate
    pH 7.0


This protocol was adapted from Hamorsky et al. (2013). The magnICON vectors were provided from Icon Genetics GmbH, Halle/Saale, Germany. This work was made possible in part by NIH R21/R33AI088585 and a University of Louisville Office of Vice President for Research Competitive Enhancement Grant (50730) to NM.


  1. Giritch, A., Marillonnet, S., Engler, C., van Eldik, G., Botterman, J., Klimyuk, V. and Gleba, Y. (2006). Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors. Proc Natl Acad Sci U S A 103(40): 14701-14706. 
  2. Hamorsky, K. T., Grooms-Williams, T. W., Husk, A. S., Bennett, L. J., Palmer, K. E. and Matoba, N. (2013). Efficient single tobamoviral vector-based bioproduction of broadly neutralizing anti-HIV-1 monoclonal antibody VRC01 in Nicotiana benthamiana plants and utility of VRC01 in combination microbicides. Antimicrob Agents Chemother 57(5): 2076-2086.
  3. Matoba, N., Davis, K. R. and Palmer, K. E. (2011). Recombinant protein expression in Nicotiana. Methods Mol Biol 701: 199-219.
  4. Marillonnet, S., Giritch, A., Gils, M., Kandzia, R., Klimyuk, V. and Gleba, Y. (2004). In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proc Natl Acad Sci U S A 101(18): 6852-6857.


基于植物的表达系统为重组蛋白提供了替代的生物制造平台(Matoba等人,2011)。特别地,基于植物病毒的载体可以在烟草(Nicotiana benthamiana)(本塞姆氏烟草)的叶组织中天数内过表达蛋白质。为了克服重组病毒的遗传不稳定性和有限的感染性的问题,土壤杆菌介导的"解构的"病毒载体的递送已经成为生产大和/或多组分蛋白如免疫球蛋白的主要方法Ig)G单克隆抗体(mAb)。在这里,我们描述了在N中产生人IgG mAbs的方法。本生烟草使用烟草花叶病毒复制子载体magnICON 。载体在7天内可以表达高达几百mg的mAb/kg叶材料。显示了分别用于广泛中和的抗HIV和抗流感mAbs VRC01和CR6261的代表性病例(Hamorsky等人,2013)。叶组织匀浆,通过过滤和离心澄清提取物。 mAb通过快速蛋白质液相色谱(FPLC)使用蛋白A亲和力和Phenyl HP疏水界面树脂纯化。

关键字:单克隆抗体, 烟草, 植物病毒载体


  1. 根癌土壤杆菌电感受态细胞(菌株GV3101)
  2. (Icon Genetics GmbH,目录号:pICH38099)(Marillon et al。,2004; Giritch et al。,2006)。 br />
  3. 利福平(Sigma-Aldrich,目录号:R3501)
  4. 庆大霉素(Sigma-Aldrich,目录号:G1264)
  5. 卡那霉素(Sigma-Aldrich,目录号:K1876)
  6. 酵母提取物(Fisher Scientific,目录号:BP1422)
  7. 营养肉汤(BD,目录号:231000)
  8. UV/Vis比色杯(VWR International,目录号:77776-745)
  9. MES(默克密理博公司,目录号:475894)
  10. 硫酸镁(EMD Millipore,目录号:MX0070-3)
  11. 磷酸钠(EMD Millipore,目录号:SX0710-1)
  12. 氯化钠(Sigma-Aldrich,目录号:S9888)
  13. 氢氧化钠
  14. 抗坏血酸(Fisher Scientific,目录号:BP351)
  15. 1M Tris缓冲液(pH 8.0)
  16. 甘氨酸(Fisher Scientific,目录号:BP381-5)
  17. L-精氨酸(Sigma-Aldrich,目录号:A5006)
  18. Dulbecco's磷酸盐缓冲盐水(DPBS)(Gibco ,目录号:14190-144)
  19. LB琼脂平板(见配方)
  20. YenB媒体(见配方)
  21. 渗透缓冲液(参见配方)
  22. 提取缓冲液(参见配方)
  23. 蛋白A洗脱缓冲液(参见配方)
  24. 蛋白A平衡/洗涤缓冲液(参见配方)
  25. Phenyl HP平衡/清洗缓冲液(参见配方)
  26. Phenyl HP洗脱缓冲液(见配方)


  1. Amicon超离心过滤器,30K(EMD Millipore,目录号:UFC903024)
  2. 电穿孔杯(USA Scientific,目录号:9104-1050)
  3. 瓶顶过滤器单元(VWR International,目录号:73520-986)
  4. 斜面烧瓶(VWR International,目录号:4446-500)
  5. 离心瓶(Thermo Fisher Scientific,目录号:3141-0250)
  6. HiTrap Protein A HP柱(通用电气公司,目录号:17-0403-03)
  7. Phenyl HP柱(通用电气公司,目录号:17-5195-01)
  8. Multiporator(Eppendorf,Bacteria module,目录号:4308 805.005)
  9. 28℃孵育器(Fisher Scientific)
  10. 台式轨道摇床(Thermo Fisher Scientific,目录号:SHKE4450)
  11. UV/Vis分光光度计(Beckman Coulter,目录号:DU800)
  12. Avanti J-26 XP离心机(Beckman Coulter,目录号:393124)
  13. 渗透装置(Bel-Art Products,目录号:F420250000)
  14. 真空泵(William H.Welch Medical Library,目录号:8890A-75)
  15. 生长室
  16. Blender(Waring Pro,目录号:7011HS)
  17. AKTA净化器(通用电气公司,目录号:28-4062-66)


  1. 用含有感兴趣mAb序列的植物表达病毒载体DNA质粒将电感受态农杆菌细胞在2,000V下转化5毫秒。
  2. 在28℃下振荡转化细胞1小时以建立抗生素抗性后,在LB琼脂平板(含有30μg/ml利福平,50μg/ml庆大霉素和50μg/ml卡那霉素)上划线细胞,并在28℃培养箱中生长 3 - 4天。
  3. 挑取单个转化的细菌菌落,并在5ml YenB培养基中加上30μg/ml利福平和50μg/ml卡那霉素,在28℃和225rpm下生长过夜。
  4. 将5毫升起始培养物转移到含有95毫升新鲜YenB培养基加上相同抗生素的大烧瓶中,并在28℃和225转/分钟生长过夜。
  5. 使用UV/Vis分光光度计,使用YenB介质作为空白,测量600nM的光密度。计算在所需的浸润缓冲液(1.6L)的总体积中最终OD 600等于0.03所需的大培养物的体积。
  6. 离心计算体积的细菌培养物在5,000×g,室温下15-30分钟。将沉淀的细菌重悬在任何体积的浸润缓冲液中,稀释至最终体积为1.6L 注意:例如,如果度量OD <600> 为1.25,则会进行以下计算
    (1.25)x =(0.03)(1.6 L)
    x = 38.4ml的细菌培养物需要离心,然后重新悬浮在浸润缓冲液中。
  7. 将重悬的细菌转移到真空装置中并填充至最终体积。对整个N的所有叶施加25英寸Hg的压力。本生烟草(在26-30天之间)2分钟,然后释放真空,导致细菌接种物进入叶子(图1)。相同的浸润缓冲液可以重复用于多达约16个植物,而没有任何明显的接种贬值。

    图1.真空渗透。mAb载体通过 A递送。使用真空渗透(参见上述,步骤7),将叶片上的根瘤土壤转化为本氏烟草叶。
  8. 植物在浸润后生长与浸润前相同,在27℃和> 50%湿度,16小时白天/8小时夜间时间表。每隔一天给植物浇水。
  9. 在浸润后7天(dpi),通过在茎的基部切割叶并收集感染的表达mAb的叶,并测量总质量。与未接种的叶相比,接种后的叶可通过浸润4-5天后显示的马赛克表型来区别。
  10. 使用工业混合器在冷提取缓冲液中提取mAb,同时每1g叶组织混合2ml缓冲液 注意:对于剩余的程序,请尽力确保mAb保持在4°C,包括提取和纯化。
  11. 将叶提取物在4℃下以15,000×g离心10分钟以沉淀植物碎片。
  12. 使用氢氧化钠将部分澄清的提取物的pH调节至7.0,并在4℃下以15,000×g离心10分钟以沉淀淀粉和小植物碎片。
  13. 提取的最后步骤需要使用真空使澄清的提取物通过0.2μm瓶顶过滤器。
  14. mAb最初通过使用来自GE Healthcare的HiTrap Protein A HP柱在AKTA纯化器上通过FPLC纯化。该柱用10柱体积(CV)的蛋白A平衡/洗涤缓冲液平衡
  15. mAb澄清的提取物以2ml/min加载。在将提取物加载到柱之后,使用蛋白A平衡/洗涤缓冲液(10CV)将未结合的蛋白从柱中洗出。
  16. 蛋白A洗脱缓冲液用​​于使用100mM蛋白A洗脱缓冲液的梯度梯度从蛋白A柱洗脱mAb达10个CV。使用1M Tris缓冲液(pH 8.0)将含有mAb的级分的pH从3.0调节至7.0 注意:步骤梯度被编程到方法运行中,使得AKTA纯化器在一个步骤中从0%洗脱缓冲液转换到100%洗脱缓冲液。
  17. 在蛋白A纯化后,使用来自GE Healthcare的HiTrap Phenyl HP柱。柱子在苯基HP平衡/洗涤缓冲液中用10CV平衡
  18. 蛋白A洗脱的mAb在Phenyl HP平衡/洗涤缓冲液中以1:10稀释,并以2ml/min加载。使用10 CV苯基HP平衡/洗涤缓冲液将未结合的蛋白从柱上洗去
  19. Phenyl HP洗脱缓冲液用​​于使用从0%苯基HP洗脱缓冲液到30%CV的100%苯基HP洗脱缓冲液的线性梯度从柱中洗脱mAb,然后在100%苯基HP洗脱缓冲液中另外5个CV。 > 注意:线性梯度被编程到方法运行中,使得AKTA纯化器控制从0%洗脱缓冲液到100%洗脱缓冲液的递增转变,其中洗脱的整个长度为30CV。
  20. 将纯化的mAb配制在pH 7.2的dulbecco's磷酸盐缓冲盐水(DPBS)中。通过在还原和非还原条件下过载的考马斯染色的十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)的密度测定法分析纯度。
  21. 活性通过抗原捕获酶联免疫吸附测定(ELISA),表面等离子体共振和体外病毒中和测定来确定。


  1. LB琼脂平板上 30μg/ml利福平
  2. YenB介质(1升)
    营养肉汤8.0 g
    pH 7.0
  3. 渗透缓冲液
    10 mM MES
    pH 5.5
  4. 提取缓冲区
    100 mM磷酸钠
    100 mM氯化钠
    pH 6.0
  5. 蛋白A洗脱缓冲液
    100 mM甘氨酸 200mM L-精氨酸 pH 3.0
  6. 蛋白A平衡/洗涤缓冲液
    20 mM磷酸钠
    pH 7.0
  7. Phenyl HP平衡/洗涤缓冲液
    1 M硫酸铵
    pH 7.0
  8. Phenyl HP洗脱缓冲液
    pH 7.0


该方案改编自Hamorsky等人(2013)。 magnICON载体由Icon Genetics GmbH,Halle/Saale,Germany提供。 这项工作部分由NIH R21/R33AI088585和路易斯维尔大学副校长办公室研究竞争力增强拨款(50730)到NM。


  1. Giritch,A.,Marillonnet,S.,Engler,C.,van Eldik,G.,Botterman,J.,Klimyuk,V.and Gleba,Y。(2006)。 在用非竞争性病毒载体共感染的植物中快速高产量表达全长IgG抗体。 a> Proc Natl Acad Sci USA 103(40):14701-14706。 
  2. Hamorsky,K.T.,Grooms-Williams,T.W.,Husk,A.S.,Bennett,L.J.,Palmer,K.E.and Matoba,N。(2013)。 广泛中和抗HIV-1单克隆抗体VRC01的高效率单病毒载体生物制备>本尼巴烟草(Nicotiana benthamiana)植物和VRC01在组合杀微生物剂中的用途。 Antimicrob Agents Chemother 57(5):2076-2086。
  3. Matoba,N.,Davis,K.R。和Palmer,K.E。(2011)。 Nicotiana中的重组蛋白表达 。方法Mol Biol 701:199-219。
  4. Marillonnet,S.,Giritch,A.,Gils,M.,Kandzia,R.,Klimyuk,V.and Gleba,Y。(2004)。 在植物中病毒RNA复制子的工程化:通过DNA重组的有效装配通过农杆菌递送的模块。 Proc Natl Acad Sci USA 101(18):6852-6857。
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引用:Husk, A., Hamorsky, K. T. and Matoba, N. (2014). Monoclonal Antibody Purification (Nicotiana benthamiana Plants). Bio-protocol 4(2): e1034. DOI: 10.21769/BioProtoc.1034.



zahid naseri
Greetings and Regards
How do I prepare magicon Vector ?
please guide me
7/12/2016 12:41:24 PM Reply