High-Yield Production of Receptor Binding Domain of SARS-CoV-2 Linked to Bacterial Flagellin in Plants Using Self-Replicating Viral Vector pEff

The development of recombinant vaccines against SARS-CoV-2 is required to eliminate the COVID-19 pandemic. We reported the expression of a recombinant protein Flg-RBD comprising receptor binding domain of SARS-CoV-2 spike glycoprotein (RBD) fused to flagellin of Salmonella typhimurium (Flg), known as mucosal adjuvant, in Nicotiana benthamiana plants. The fusion protein, targeted to the cytosol, was transiently expressed using the self-replicating vector pEff based on potato virus X genome. The recombinant protein Flg-RBD was expressed at the level of about 110–140 μg per gram of fresh leaf tissue and was found to be insoluble. The fusion protein was purified using metal affinity chromatography under denaturing conditions. To increase the yield of Flg-RBD, the flow-through fraction obtained after loading of the protein sample on the Ni-NTA resin was re-loaded on the sorbent. The yield of Flg-RBD after purification reached about 100 μg per gram of fresh leaf tissue and the purified protein remained soluble after dialysis. The control flagellin was expressed in a soluble form and its yield after purification was about 300 μg per gram of fresh leaf biomass. Plant-produced Flg-RBD protein could be further used for the development of intranasal recombinant mucosal vaccines against COVID-19.