Monoclonal Antibody Purification (Nicotiana benthamiana Plants)
单克隆抗体的纯化（本氏烟草）
发布: 2014年01月20日第4卷第2期 DOI: 10.21769/BioProtoc.1034 浏览次数: 14837
评审: Pinchas TsukermanAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
May 2013
Abstract
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
- Agrobacterium tumefaciens electrocompetent cells (strain GV3101)
- MagnICON® plasmid (Icon Genetics GmbH, catalog number: pICH38099 ) (Marillonnet et al., 2004; Giritch et al., 2006)
- Rifampicin (Sigma-Aldrich, catalog number: R3501 )
- Gentamicin (Sigma-Aldrich, catalog number: G1264 )
- Kanamycin (Sigma-Aldrich, catalog number: K1876 )
- Yeast Extract (Fisher Scientific, catalog number: BP1422 )
- Nutrient Broth (BD, catalog number: 231000 )
- UV/Vis cuvette (VWR International, catalog number: 77776-745 )
- MES (EMD Millipore, catalog number: 475894 )
- Magnesium sulfate (EMD Millipore, catalog number: MX0070-3 )
- Sodium phosphate (EMD Millipore, catalog number: SX0710-1 )
- Sodium chloride (Sigma-Aldrich, catalog number: S9888 )
- Sodium hydroxide
- Ascorbic acid (Fisher Scientific, catalog number: BP351 )
- 1M Tris buffer (pH 8.0)
- Glycine (Fisher Scientific, catalog number: BP381-5 )
- L-Arginine (Sigma-Aldrich, catalog number: A5006 )
- Dulbecco’s Phosphate Buffered Saline (DPBS) (Gibco®, catalog number: 14190-144 )
- LB agar plate (see Recipes)
- YenB media (see Recipes)
- Infiltration buffer (see Recipes)
- Extraction buffer (see Recipes)
- Protein A Elution buffer (see Recipes)
- Protein A Equilibration/Wash buffer (see Recipes)
- Phenyl HP Equilibration/Wash buffer (see Recipes)
- Phenyl HP Elution buffer (see Recipes )
Equipment
- Amicon Ultra centrifugal filter, 30 K (EMD Millipore, catalog number: UFC903024 )
- Electroporation cuvette (USA Scientific, catalog number: 9104-1050 )
- Bottle top filter unit (VWR International, catalog number: 73520-986 )
- Beveled flask (VWR International, catalog number: 4446-500 )
- Centrifuge bottles (Thermo Fisher Scientific, catalog number: 3141-0250 )
- HiTrap Protein A HP column (General Electric Company, catalog number: 17-0403-03 )
- Phenyl HP column (General Electric Company, catalog number: 17-5195-01 )
- Multiporator (Eppendorf, Bacteria module, catalog number: 4308 805.005 )
- 28 °C Incubator (Fisher Scientific)
- Benchtop orbital shaker (Thermo Fisher Scientific, catalog number: SHKE4450 )
- UV/Vis spectrophotometer (Beckman Coulter, catalog number: DU800 )
- Avanti® J-26 XP Centrifuge (Beckman Coulter, catalog number: 393124 )
- Infiltration apparatus (Bel-Art Products, catalog number: F420250000 )
- Vacuum pump (William H. Welch Medical Library, catalog number: 8890A-75 )
- Growth chamber
- Blender (Waring Pro, catalog number: 7011HS )
- AKTA purifier (General Electric Company, catalog number: 28-4062-66 )
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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.
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分类
植物科学 > 植物生物化学 > 蛋白质
生物化学 > 蛋白质 > 分离和纯化
免疫学 > 抗体分析 > 抗体检测
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