Fig 2: A Western blot analysis of human ACE2s, produced in Nicotiana benthamiana plants. dACE2: angiotensin-converting enzyme 2 (ACE2) co-expressed with bacterial Endo H, produced in N. benthamiana, different concentration (dilutions) of crude extract were loaded into wells; gACE2: western blot analysis of human ACE2, produced in N. benthamiana plants; different concentration (dilutions) of crude extract were loaded into wells; C-undiluted crude extract from non-infiltrated N. benthamiana, was loaded into well; gPA83: 25, 50, and 100 ng of purified plant produced dPA83 of Bacillus anthracis, loaded as a control protein to quantify the expression levels of ACE2 and ACE2 proteins. Purified anti-His Tag antibody (Cat. No. 652502, BioLegend) was used as primary and mouse IgG used as secondary antibodies to detect ACE2 proteins.

Fig 3: Binding activity of plant produced glycosylated or deglycosylated variants of ACE2 with commercial or plant produced, glycosylated or deglycosylated forms of spike proteins (Flag tagged). Commercial or plant-produced spike protein was coated with an ELISA plate at a concentration of 200 ng/well. Different concentration of plant produced, Sephacryl® S-200 HR column purified, gACE2 or dACE2 proteins (His-tagged) were added. Purified anti-human monoclonal ACE2 antibody (Cat. No. 375801, BioLegend) was used as a primary and rat IgG used as secondary antibodies. Com S: commercial Spike protein, active Recombinant 2019-nCoV Spike Protein, RBD, His Tag, produced in Baculovirus-Insect Cells, Cat. no. MBS2563882); pp-gRBD: plant produced glycosylated Receptor Binding Domain of Spike protein (Mamedov et al., 2021); pp-dRBD: plant produced deglycosylated RBD (Mamedov et al., 2021); pp-gACE2: plant produced gACE2; pp-dACE2: plant produced Endo H in vivo dACE2; Endo H, plant produced Flag-tagged protein was used as negative control. (A,B) Graph for binding affinity between pp-gACE2 and pp-dACE2 to spike protein variants. (A) A graph was plotted with non-linear regression analysis in GraphPad software. Points refer to absorbance for each sample dilutions and lines were plotted according to Kd value. (B) Column bar graph of Kd values determined with non-linear regression analysis in GraphPad software.

Fig 4: Apparent activities of two distinct ACE2 derivatives produced in plants to RBDs plotted against half maximal inhibitory concentration (IC50) of authentic SARS-CoV-2 neutralization. The IC50 values of the gACE2 (glycosylated) and dACE2 (deglycosylated) were calculated using normalized optical density (OD) data obtained from quadruplicated test dilutions in GraphPad Prism v8.2 software (GraphPad). The OD values from untreated (cell control) wells were used as normalization standards. A non-linear regression analysis was performed using log (inhibitor) versus normalized response-variable slope. The R square values were recorded as 0.86 and 0.89 for dACE2 and ACE2, respectively. ACE2, gACE2, glycosylated ACE2; dACE2, plant produced deglycosylated ACE2.

Fig 5: Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (A) and Western blot (B,C) analysis of plant produced, Ni-NTA resin purified gACE2 or dACE2 proteins. Glycosylated and deglycosylated plant produced ACE2 proteins were purified from N. benthamiana plant using HisPur™ Ni-NTA resin. gACE2: 5 or 10 μg purified gACE2 protein was loaded into well; dACE2: 5 or 10 μg purified dACE2 proteins were loaded into well. Bovine serum albumin (BSA) standards: 1.0, 2.5, and 5.0 μg BSA protein was loaded as a standard protein. (B) Membrane was probed with anti-His6 antibody. gPA83 (plant produced glycosylated protective antigen of B. anthracis, MM ∼100 kDa) and dPA83 (deglycosylated protective antigen of B. anthracis, MM ∼90 kDa) proteins were used as a standard. (C) Membrane was probed with a purified anti-human monoclonal ACE2 antibody (Cat. No. 375801, BioLegend, CA, United States). cACE2: recombinant human ACE2, amino acid (Gln18—Ser740) (Accession: NM_021804.1) was expressed in CHO cells as His tagged protein (cat. no. 792004, BioLegend, CA, United States). The image was taken using a highly sensitive GeneGnome XRQ Chemiluminescence imaging system.