Expression optimization, purification and in vitro characterization of human epidermal growth factor produced in Nicotiana benthamiana

Hanittinan, Oranicha; Oo, Yamin; Chaotham, Chatchai; Rattanapisit, Kaewta; Shanmugaraj, Balamurugan; Phoolcharoen, Waranyoo

doi:10.1016/j.btre.2020.e00524

Cited by 24 publications

(24 citation statements)

References 49 publications

(53 reference statements)

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“…Because the investment cost and time for phase 1 cGMP manufacturing of plant expression system are comparatively cost-effective (7.5- to 10-fold) and faster (<6 months) than mammalian expression system, the plant-production platform might be an alternative platform for the production of many biopharmaceutical products. The use of plant expression systems for the production of pharmaceutically important proteins ( Bulaon et al, 2020 ; Hanittinan et al, 2020 ), vaccines ( Marsian et al, 2017 ; Rosales-Mendoza et al, 2017 ), diagnostic reagents ( Rattanapisit et al, 2021 ), and antibodies ( Kopertekh et al, 2019 ; Hurtado et al, 2020 ; Rattanapisit et al, 2020 ) were documented. In addition, the prior studies reported that the generation of plant-made protective immunogen and therapeutic antibodies ( Dent et al, 2016 ; Rattanapisit et al, 2019a , b ; Nessa et al, 2020 ; Yiemchavee et al, 2021 ).…”

Section: Discussionmentioning

confidence: 99%

Functional Characterization of Pembrolizumab Produced in Nicotiana benthamiana Using a Rapid Transient Expression System

et al. 2021

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The striking innovation and clinical success of immune checkpoint inhibitors (ICIs) have undoubtedly contributed to a breakthrough in cancer immunotherapy. Generally, ICIs produced in mammalian cells requires high investment, production costs, and involves time consuming procedures. Recently, the plants are considered as an emerging protein production platform due to its cost-effectiveness and rapidity for the production of recombinant biopharmaceuticals. This study explored the potential of plant-based system to produce an anti-human PD-1 monoclonal antibody (mAb), Pembrolizumab, in Nicotiana benthamiana. The transient expression of this mAb in wild-type N. benthamiana accumulated up to 344.12 ± 98.23 μg/g fresh leaf weight after 4 days of agroinfiltration. The physicochemical and functional characteristics of plant-produced Pembrolizumab were compared to mammalian cell-produced commercial Pembrolizumab (Keytruda®). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis results demonstrated that the plant-produced Pembrolizumab has the expected molecular weight and is comparable with the Keytruda®. Structural characterization also confirmed that both antibodies have no protein aggregation and similar secondary and tertiary structures. Furthermore, the plant-produced Pembrolizumab displayed no differences in its binding efficacy to PD-1 protein and inhibitory activity between programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) interaction with the Keytruda®. In vitro efficacy for T cell activation demonstrated that the plant-produced Pembrolizumab could induce IL-2 and IFN-γ production. Hence, this proof-of-concept study showed that the plant-production platform can be utilized for the rapid production of functional mAbs for immunotherapy.

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Section: Discussionmentioning

confidence: 99%

Functional Characterization of Pembrolizumab Produced in Nicotiana benthamiana Using a Rapid Transient Expression System

et al. 2021

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“…Plant expression systems have several advantages over other expression systems currently in use, such as rapid and high production with the ability to accumulate grams of target protein per kilogram of biomass in less than a week ( Gleba et al, 2005 ; Sainsbury et al, 2009 ; Holtz et al, 2015 ; Maharjan and Choe, 2021 ). This system has been successfully used for the production of functional active complex proteins, such as full length Pfs48/45 of Plasmodium falciparum ( Mamedov et al, 2019b ), human Furin, Factor IX ( Mamedov et al, 2019a ), glycohormone erythropoietin ( Grosse-Holz et al, 2018 ), latent transforming growth factor-beta (TGF-beta) ( Wilbers et al, 2016 ), human epidermal growth factor ( Hanittinan et al, 2020 ), PA83 of B. anthracis ( Mamedov et al, 2016 , 2017 ), HIV gp140, other viral glycoproteins ( Margolin et al, 2018 , 2020a ), and antigens against influenza, Ebola, dengue, rotavirus, and norovirus ( Mett et al, 2008 ; Landry et al, 2014 ; Rybicki, 2014 ; Pêra et al, 2015 ; Takeyama et al, 2015 ). The goal of this study is to lead to the development of a safe, cost effective plant produced ACE2 enzyme with anti-SARS-CoV-2 activities to use in the treatment of COVID-19.…”

Section: Discussionmentioning

confidence: 99%

“…The targeting of the protein to the ER provided a high level of production of a number of complex and difficult to-express proteins in N. benthamiana plant, such as full length Pfs48/45 or Pfs230 of P. falciparum ( Farrance et al, 2011 ; Mamedov et al, 2019b ), heptameric form of protective antigen of B. anthracis , human Furin and Factor IX ( Mamedov et al, 2019a ), and RBD of spike protein of SARS-CoV-2 ( Mamedov et al, 2021 ); human Interleukin 6 ( Nausch et al, 2012 ); hemagglutinin from a range influenza viruses ( Chichester et al, 2009 ); monoclonal antibodies ( Vézina et al, 2009 ; Holtz et al, 2015 ); HIV gp140 ( Margolin et al, 2020a ). In addition, other factors that can provide more target accumulation are Agro optimization ( Shamloul et al, 2014 ; Hanittinan et al, 2020 ; Kaur et al, 2021 ), especially using appropriate target specific Agrobacterium strains ( Norkunas et al, 2018 ), activation of agrobacteria with MES (2-morpholinoethanesulfonic acid), MgCl 2 , Acetosyringone ( Shamloul et al, 2014 ; Kaur et al, 2021 ), and to control humidity, temperature, and light during plant growth ( Shamloul et al, 2014 ). In general, these factors allowed the production of the recombinant soluble form of ACE2 at a high level in the N. benthamiana plant.…”

Section: Discussionmentioning

confidence: 99%

Soluble Human Angiotensin- Converting Enzyme 2 as a Potential Therapeutic Tool for COVID-19 is Produced at High Levels In Nicotiana benthamiana Plant With Potent Anti-SARS-CoV-2 Activity

et al. 2021

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The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread to more than 222 countries and has put global public health at high risk. The world urgently needs a safe, cost-effective SARS-CoV-2 vaccine as well as therapeutic and antiviral drugs to combat COVID-19. Angiotensin-converting enzyme 2 (ACE2), as a key receptor for SARS-CoV-2 infections, has been proposed as a potential therapeutic tool in patients with COVID-19. In this study, we report a high-level production (about ∼0.75 g/kg leaf biomass) of human soluble (truncated) ACE2 in the Nicotiana benthamiana plant. After the Ni-NTA single-step, the purification yields of recombinant plant produced ACE2 protein in glycosylated and deglycosylated forms calculated as ∼0.4 and 0.5 g/kg leaf biomass, respectively. The plant produced recombinant human soluble ACE2s successfully bind to the SARS-CoV-2 spike protein. Importantly, both deglycosylated and glycosylated forms of ACE2 are stable at increased temperatures for extended periods of time and demonstrated strong anti-SARS-CoV-2 activities in vitro. The half maximal inhibitory concentration (IC50) values of glycosylated ACE2 (gACE2) and deglycosylated ACE2 (dACE2) were ∼1.0 and 8.48 μg/ml, respectively, for the pre-entry infection, when incubated with 100TCID50 of SARS-CoV-2. Therefore, plant produced soluble ACE2s are promising cost-effective and safe candidates as a potential therapeutic tool in the treatment of patients with COVID-19.

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“…Our results demonstrated that recombinant GCase was successfully produced in WT and ΔgntI N. benthamiana plants ( Figure 1 ). Since Agrobacterium -infiltration conditions usually affect the transformation efficiency, resulting in different expression levels of recombinant protein (Hanittinan et al, 2020 ), two main factors, Agrobacterium cell density and harvest time, were optimized to produce a large amount of GCase with biological activity. Previous studies demonstrated that too low cell density might lead to insufficient Agrobacterium infection, whereas too high density may cause necrosis (Kagale et al, 2012 ; Leuzinger et al, 2013 ; Yan et al, 2019 ).…”

Section: Discussionmentioning

confidence: 99%

Transient Production of Human β-Glucocerebrosidase With Mannosidic-Type N-Glycan Structure in Glycoengineered Nicotiana benthamiana Plants

et al. 2021

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Gaucher disease is an inherited lysosomal storage disorder caused by a deficiency of functional enzyme β-glucocerebrosidase (GCase). Recombinant GCase has been used in enzyme replacement therapy to treat Gaucher disease. Importantly, the terminal mannose N-glycan structure is essential for the uptake of recombinant GCase into macrophages via the mannose receptor. In this research, recombinant GCase was produced using Agrobacterium-mediated transient expression in both wild-type (WT) and N-acetylglucosaminyltransferase I (GnTI) downregulated Nicotiana benthamiana (ΔgntI) plants, the latter of which accumulates mannosidic-type N-glycan structures. The successfully produced functional GCase exhibited GCase enzyme activity. The enzyme activity was the same as that of the conventional mammalian-derived GCase. Notably, N-glycan analysis revealed that a mannosidic-type N-glycan structure lacking plant-specific N-glycans (β1,2-xylose and α1,3-fucose residues) was predominant in all glycosylation sites of purified GCase produced from ΔgntI plants. Our research provides a promising alternative plant line as a host for the production of recombinant GCase with a mannosidic-type N-glycan structure. This glycoengineered plant might be applicable to the production of other pharmaceutical proteins, especially mannose receptor targeted protein, for therapeutic uses.

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Expression optimization, purification and in vitro characterization of human epidermal growth factor produced in Nicotiana benthamiana

Cited by 24 publications

References 49 publications

Functional Characterization of Pembrolizumab Produced in Nicotiana benthamiana Using a Rapid Transient Expression System

Functional Characterization of Pembrolizumab Produced in Nicotiana benthamiana Using a Rapid Transient Expression System

Soluble Human Angiotensin- Converting Enzyme 2 as a Potential Therapeutic Tool for COVID-19 is Produced at High Levels In Nicotiana benthamiana Plant With Potent Anti-SARS-CoV-2 Activity

Transient Production of Human β-Glucocerebrosidase With Mannosidic-Type N-Glycan Structure in Glycoengineered Nicotiana benthamiana Plants

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