Production of the SARS-CoV-2 Spike protein and its Receptor Binding Domain in plant cell suspension cultures

Abstract

The COVID-19 pandemic, caused by the worldwide spread of SARS-CoV-2, has prompted the scientific community to rapidly develop efficient and specific diagnostics and therapeutics. A number of avenues have been explored, including the manufacture of COVID-related proteins to be used as reagents for diagnostics or treatment. The production of RBD and Spike proteins was previously achieved in eukaryotic cells, mainly mammalian cell cultures, while the production in microbial systems has been unsuccessful until now. Here we report the effective production of SARS-CoV-2 proteins in two plant model systems. We established transgenic tobacco BY-2 and Medicago truncatula A17 cell suspension cultures stably producing the full-length Spike and RBD recombinant proteins. For both proteins, various glycoforms were obtained, with higher yields in Medicago cultures than BY-2. This work highlights that RBD and Spike can be secreted into the culture medium, which will impact subsequent purification and downstream processing costs. Analysis of the culture media indicated the presence of the high molecular weight Spike protein of SARS-CoV-2. Although the production yields still need improvement to compete with mammalian systems, this is the first report showing that plant cell suspension cultures are able to produce the high molecular weight Spike protein. This finding strengthens the potential of plant cell cultures as production platforms for large complex proteins.

A simplified protocol for Agrobacterium-mediated transformation of cell suspension cultures of the model species Medicago truncatula A17

“…To validate the effectiveness of the new transformation protocol, we selected a recombinant protein with a high impact in current worldwide research, the Receptor Binding Domain of the SARS-CoV-2 virus (Rebelo et al 2022 ). Other ongoing projects in the laboratory, in which we have used this new protocol, indicate that it is faster and simpler than previous methods (Rebelo et al in preparation; Vieira et al in preparation).…”

“…In this study we used the Receptor Binding Domain (RBD) of the SARS-CoV-2 coronavirus sequence codon optimized for Nicotiana tabacum and cloned into pTRA vector (pTRA_RBD) as described in Rebelo et al ( 2022 ). A. tumefaciens GV3101::PMP90RK containing pTRA_RBD was grown in YEB medium supplemented with 2 mM Mg 2 SO 4 , 50 mg/L Rifampicin and Kanamycin and 80 mg/L Carbenicillin (antibiotics from NZYTech).…”

Plants as Biofactories for Therapeutic Proteins and Antiviral Compounds to Combat COVID-19

“…In order to increase the immunogenicity of the antigen, RBD fused to flagellin of Salmonella typhimurium (Flg), known as mucosal adjuvant, was also transiently expressed with N. benthamiana using a self-replicating viral vector [ 76 ]. As an alternative to the transient expression platform, tobacco BY-2 and Medicago truncatula A17 cell suspension cultures were also used to stably express both full-length S protein and RBD [ 73 ]. The results showed that recombinant S protein and RBD could be secreted into the culture medium, which facilitated the subsequent purification and reduced the downstream processing costs.…”

Performance of plant-produced RBDs as SARS-CoV-2 diagnostic reagents: a tale of two plant platforms

“…Plant-based recombinant COVID-19 works have mostly been based on transient expression in Nicotiana benthamiana (see Table 1 and references therein). Only few studies involved the stable transformation of plant cell suspensions ( Daniell et al., 2022 ; Rebelo et al., 2022 ; Ferreira et al., 2023 ; Shen et al., 2023 ). Among the proteins reported, there are structural proteins such as the spike (S) protein or S subunits (S1 and S2), the receptor-binding domain (RBD), the nucleocapsid (NC), envelope (E), and membrane (M) viral proteins, and others such as angiotensin-converting enzyme 2 (ACE2) receptor and antibodies against domains of the spike protein (RBD and S1) and the nucleocapsid protein ( Table 1 ).…”