Production of ORF8 protein from SARS-CoV-2 using an inducible virus-mediated expression-system in suspension-cultured tobacco BY-2 cells

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which spread worldwide in 2019, is an urgent problem to be overcome. The ORF8 of SARS-CoV-2 has been suggested to be associated with the symptoms of COVID-19, according to reports of clinical studies. However, little is known about the function of ORF8. As one of the ways to advance the functional analysis of ORF8, mass production of ORF8 with the correct three-dimensional structure is necessary. In this study, we attempted to produce ORF8 protein by chemical-inducible protein production system using tobacco BY-2 cells. An ORF8-producing line was generated by the Agrobacterium method. As a result, the production of ORF8 of 8.8 {+/-} 1.4 mg/L of culture medium was confirmed. SDS-PAGE and Nuclear magnetic resonance (NMR) analysis confirmed that the ORF8 produced by this system is a dimeric form with a three-dimensional structure, unlike that produced in Escherichia coli. Furthermore, it was suggested that the ORF8 produced by this system was N-glycosylated. Through this study, we succeeded in producing ORF8 with the correct three-dimensional structure in a chemical-inducible protein production system using tobacco BY2 cells. It is expected that the functional analysis of ORF8 will be advanced using the ORF8 produced by this system and that it will greatly contribute to the development of vaccines and antibodies against ORF8.