SARS-CoV-2 Fusion Protein Expression

ORFs coding for Nef^mut fused with S1, S2, M, or N SARS-CoV-2 proteins were cloned into pVAX1 plasmid (Thermo Fisher, Waltham, MA, USA), i.e., a vector approved by FDA for use in humans. To obtain the pVAX1 vector expressing Nef^mut, its ORF was cloned into Nhe I and EcoR I sites. To recover vectors expressing Nef^mut-based fusion products, an intermediate vector referred to as pVAX1/Nef^mutfusion was produced. Here, the whole Nef^mut ORF deprived of its stop codon was followed by a sequence coding a GPGP linker including a unique Apa I restriction site. In this way, sequences comprising the Apa I site at their 5′ end, and the Pme I one at their 3′ end were fused in frame with Nef^mut ORF. Stop codons of SARS-CoV-2-related sequences were preceded by sequences coding for a DYKDDDK epitope tag (flag-tag). SARS-CoV-2 sequences were optimized for expression in human cells through GeneSmart software from Genescript. All these vectors were synthesized by Explora Biotech. The pTargeT vector expressing the Nef^mut/HPV16-E7 fusion protein was already described [38 (link)].