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. 2022 Dec 14;17(12):e0277668. doi: 10.1371/journal.pone.0277668

Fig 1. Expression and purification of SARS-CoV-2 RBD in Nicotiana benthamiana.

Fig 1

(A) Agro-infiltrated Nicotiana benthamiana growing in greenhouse. (B) Schematic representation of genetic construct used to express SARS-CoV-2 RBD in planta. The SARS-CoV-2 sequence was expressed as a recombinant protein with a dual 8xHis and Twin-Strep II tag, interspersed with Gly-Ser linkers (gold boxes). An ER-retention KDEL sequence was positioned at the C-terminus, and a Thrombin cleavage site (LVPRGS) was included for tag removal. (C) Left panel: Anti-His IB of samples obtained from N. benthamiana 2 to 5 days post-infiltration (dpi) with RBD construct in B. Loading control at 5 dpi reproducibly demonstrates reduced abundance of protein due to initiation of tissue necrosis at this time. Right panel: NT control (lane 1) compared to RBD expressed in N. benthamiana with calreticulin (lane 2). Anti-his IB. (D) Co-infiltration of human calreticulin (CRT) increases expression levels of RBD in N. benthamiana. Samples collected 4dpi. Anti-his IB. (E) Anti-S1 IB of purified RBD expressed in N. benthamiana (lane 1) and control RBD expressed in mammalian 293F cells (lane 2). Arrow indicates expected migration of a protein corresponding to 31.3 kDa. (F) CBB-stained SDS-PAGE of purified RBD expressed in N. benthamiana (lane 1) and control RBD expressed in mammalian 293F cells (lane 2). (G) CBB-stained SDS-PAGE of plant-derived RBD treated with (+) and without (-) the amidase Peptide-N-Glycosidase F (PNGaseF), an enzyme that cleaves N-linked glycan chains. SP, signal peptide. IB, immunoblot. CBB, Coomassie Brilliant Blue. NT, non-transformed.