TY - JOUR
T1 - Production of igg fusion proteins transiently expressed in nicotiana benthamiana
AU - Kamzina, Aigerim S.
AU - Dipalma, Michelle P.
AU - Hunter, Joseph G.L.
AU - Diamos, Andrew G.
AU - Armer, Boyd
AU - Mor, Tsafrir S.
AU - Mason, Hugh S.
N1 - Funding Information:
We thank Maria Pia DiPalma for editing the video. We also thank the Office of Educational Outreach and Student Services at Arizona State University for their generous publication fee assistance. Research for this protocol was supported by the School of Life Sciences, Arizona State University.
Publisher Copyright:
© 2021 JoVE Journal of Visualized Experiments.
PY - 2021/1
Y1 - 2021/1
N2 - High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment's purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.
AB - High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment's purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.
UR - http://www.scopus.com/inward/record.url?scp=85099953695&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85099953695&partnerID=8YFLogxK
U2 - 10.3791/61774
DO - 10.3791/61774
M3 - Article
C2 - 33522504
AN - SCOPUS:85099953695
SN - 1940-087X
VL - 2021
SP - 1
EP - 24
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
IS - 167
M1 - e61774
ER -