Monoclonal Antibody Purification (Nicotiana benthamiana Plants)
| Autor: | Krystal Teasley Hamorsky, Nobuyuki Matoba, Adam S. Husk |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Infectivity
biology medicine.drug_class viruses Strategy and Management Mechanical Engineering fungi Metals and Alloys food and beverages Nicotiana benthamiana Fast protein liquid chromatography biology.organism_classification Monoclonal antibody Molecular biology Industrial and Manufacturing Engineering law.invention CR6261 law Plant virus biology.protein Recombinant DNA medicine Protein A |
| Zdroj: | BIO-PROTOCOL. 4 |
| ISSN: | 2331-8325 |
| DOI: | 10.21769/bioprotoc.1034 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
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