Abstract
Nicotiana benthamiana is used worldwide as production host for recombinant proteins. Many recombinant proteins such as monoclonal antibodies, growth factors or viral antigens require posttranslational modifications like glycosylation for their function. Here, we transiently expressed different variants of the glycosylated receptor binding domain (RBD) from the SARS-CoV-2 spike protein in N. benthamiana. We characterized the impact of variations in RBD-length and posttranslational modifications on protein expression, yield and functionality. We found that a truncated RBD variant (RBD-215) consisting of amino acids Arg319-Leu533 can be efficiently expressed as a secreted soluble protein. Purified RBD-215 was mainly present as a monomer and showed binding to the conformation-dependent antibody CR3022, the cellular receptor angiotensin converting enzyme 2 (ACE2) and to antibodies present in convalescent sera. Expression of RBD-215 in glycoengineered ΔXT/FT plants resulted in the generation of complex N-glycans on both N-glycosylation sites. While site-directed mutagenesis showed that the N-glycans are important for proper RBD folding, differences in N-glycan processing had no effect on protein expression and function.
Highlights
The ongoing COVID-19 pandemic underscores the urgency to increase the preparedness for future virus outbreaks and to establish countermeasures such as platform technologies to produce recombinant proteins for subunit vaccines or viral antigens for diagnostic tests (Amanat et al, 2020)
In N. benthamiana, overexpression of human CRT increased the overall yield of several recombinant viral glycoproteins, including HIV-1 Env (Margolin et al, 2020a) and the ectodomain of the SARS-CoV2 spike protein (Margolin et al, 2020c) and attenuated ER stress responses associated with viral glycoprotein expression
The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (Figure 1A and Supplementary Figure 1) (Amanat et al, 2020; Lan et al, 2020) fused to the barley α-amylase signal peptide and a C-terminal polyhistidine tag was transiently expressed in leaves of N. benthamiana wild-type and glycoengineered XT/FT plants (Strasser et al, 2008)
Summary
The ongoing COVID-19 pandemic underscores the urgency to increase the preparedness for future virus outbreaks and to establish countermeasures such as platform technologies to produce recombinant proteins for subunit vaccines or viral antigens for diagnostic tests (Amanat et al, 2020). In N. benthamiana, overexpression of human CRT increased the overall yield of several recombinant viral glycoproteins, including HIV-1 Env (Margolin et al, 2020a) and the ectodomain of the SARS-CoV2 spike protein (Margolin et al, 2020c) and attenuated ER stress responses associated with viral glycoprotein expression. These findings underscore the importance of N-glycosylation and N-glycan-dependent quality control processes for recombinant protein production and reveal limitations that have to be addressed to make plant-based expression platforms such as N. benthamiana more attractive for economic production (Dicker and Strasser, 2015). Our data show that N-glycans on the RBD from the SARS-CoV-2 spike protein are important for protein folding and efficient RBD production as functional protein
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