Published: Vol 4, Iss 2, Jan 20, 2014 DOI: 10.21769/BioProtoc.1034 Views: 14514
Reviewed by: Pinchas TsukermanAnonymous reviewer(s)
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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 antibodyMaterials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
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.
References
Article Information
Copyright
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
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.
Category
Plant Science > Plant biochemistry > Protein
Biochemistry > Protein > Isolation and purification
Immunology > Antibody analysis > Antibody detection
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