Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the ongoing global outbreak of coronavirus disease (COVID-19) which is a significant threat to global public health. The rapid spread of COVID-19 necessitates the development of cost-effective technology platforms for the production of vaccines, drugs, and protein reagents for appropriate disease diagnosis and treatment. In this study, we explored the possibility of producing the receptor binding domain (RBD) of SARS-CoV-2 and an anti-SARS-CoV monoclonal antibody (mAb) CR3022 in Nicotiana benthamiana. Both RBD and mAb CR3022 were transiently produced with the highest expression level of 8 μg/g and 130 μg/g leaf fresh weight respectively at 3 days post-infiltration. The plant-produced RBD exhibited specific binding to the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2). Furthermore, the plant-produced mAb CR3022 binds to SARS-CoV-2, but fails to neutralize the virus in vitro. This is the first report showing the production of anti-SARS-CoV-2 RBD and mAb CR3022 in plants. Overall these findings provide a proof-of-concept for using plants as an expression system for the production of SARS-CoV-2 antigens and antibodies or similar other diagnostic reagents against SARS-CoV-2 rapidly, especially during epidemic or pandemic situation.
Highlights Growth factors play a crucial role in tissue repair and wound healing. Recombinant human epidermal growth factor was produced in N. benthamiana by agroinfiltration. Expression conditions were optimized and the recombinant protein was purified. Plant-produced hEGF facilitate the HaCaT cell proliferation and cell migration in vitro. Plant-produced hEGF can potentially be exploited for their application in tissue engineering.
Severe acute respiratory syndrome coronavirus-2 is responsible for an ongoing global outbreak of coronavirus disease (COVID-19) and represents a significant public health threat. The rapid spread of COVID-19 necessitates the development of cost-effective technology platforms for the production of diagnostic reagents/biopharmaceuticals for COVID-19. We explored the possibility of producing an anti-SARS-CoV monoclonal antibody (mAb) CR3022 and the receptor binding domain (RBD) of SARS-CoV-2 in Nicotiana benthamiana. Both RBD and the mAb were transiently expressed with the expression of 8μg/g and 130μg/g leaf fresh weight respectively. The plant-purified mAb binds to SARS-CoV-2, but fails to neutralize it in vitro. This is the first report showing the functional characterization of an anti- SARS-CoV mAb CR3022 in plants. Overall these findings showed that plants are a promising platform to produce anti-SARS-CoV mAb to use as a research reagent or a biotherapeutic in a cost-effective manner, which is especially important to developing economies during epidemics.
Omalizumab, the anti-immunoglobulin IgE antibody is the only approved and available monoclonal antibody as an auxiliary medicament for the severe respiratory allergic reactions. It forms small size immune complexes by binding to free IgE, thereby inhibiting the interaction of IgE with its receptors. Additionally, the anti-IgE can also differently shape the airflow by impeding the stimulation of IgE receptors present on structural cells in the respiratory tract. The present study aimed to use plants as an expression system for anti-human IgE antibody production, using Nicotiana benthamiana as hosts. Recombinant Agrobacterium tumefaciens containing heavy chain (HC) and light chain (LC) domains of anti-human IgE were co-transformed in N. benthamiana. The assembling of the antibody and its expression was detected by SDS-PAGE and Western blot analysis. The functional ability of the anti-IgE antibody was determined via its binding capacity with target IgE by ELISA and the inhibition of basophil activation. The anti-human IgE mAb generated in plants was shown to be effective in binding to its target IgE and inhibit the IgE-crosslink in RS-ATL8 reporter cells. Although, antibody yield and purification process have to be further optimized, this study demonstrates the use of plant expression system as a promising platform for the production of Omalizumab which showed a comparable in vitro function to that of commercial Omalizumab (Xolair) in the inhibition of basophil activation.
Human epidermal growth factor (hEGF) is a short polypeptide that has gained clinical importance in the recent decade for wound healing. Currently, plant-based expression system is considered as an alternative viable platform for low-cost recombinant protein production. Hence, this study aims to produce hEGF in Nicotiana benthamiana plants via., transient expression using a geminiviral vector. The hEGF gene constructs were designed into six different constructs. The optimal expression was observed from the construct targeting the endoplasmic reticulum with C-terminal histidine tag at the yield level up to 15.695 µg/g LFW or 0.499% TSP. The plant-produced hEGF was purified by using Nickel affinity chromatography and confirmed its identity by SDS-PAGE and Western blot probed with anti-hEGF antibody. Furthermore, the preliminary study of the protein purification efficiency was found that the high extraction volume allows better hEGF recovery and extraction buffer pH 4 could largely remove host cell proteins (HCP), especially RuBisCO. However, ammonium sulfate precipitation is inapplicable for removing HCP from plant-produced hEGF. Furthermore, the study showed that no cytotoxic effects have been found in HaCaT cells from the plant-produced hEGF which is equivalent to commercial hEGF in HaCaT cells. Hence, this study supports that the potential of plant expression system offers a simple and cost-effective approach for the industrial-scale production of recombinant hEGF.
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