RBD Cloning

The codon-optimized DNA sequence coding for the SARS-CoV-2 RBD (amino acids 319-541 from {"type":"entrez-protein","attrs":{"text":"P0DTC2","term_id":"1835922048","term_text":"P0DTC2"}}P0DTC2 – SPIKE_SARS2) from the first human isolate Wuhan-1 fused to the α-amylase signal peptide and a C-terminal hexahistidine tag was synthesized by GeneArt (Thermo Fisher Scientific). The DNA fragment was amplified by PCR with STRINGS-7F/STRINGS-8R (Supplementary Table 1), AgeI/XhoI digested and ligated into AgeI/XhoI digested plant expression vector pEAQ-HT (Sainsbury et al., 2009). To generate the pEAQ-RBD-KDEL expression vector, the sequence was amplified by PCR from the synthetic RBD DNA fragment with STRINGS-7F and RBD-6R and cloned into AgeI/XhoI digested pEAQ-HT. pEAQ-RBD-C538A was generated by cloning of the PCR product obtained with STRINGS-7F/RBD-7R into AgeI/XhoI sites of pEAQ-HT. The sequence coding for RBD-215 (amino acids 319-533) was amplified from the synthetic DNA using STRINGS-7F/RBD-9R and cloned into pEAQ-HT. To express the RBD-215 mutants RBD-215-1Q, RBD-215-2Q and RBD-215-12Q (Supplementary Figure 1), synthetic RBD DNA fragments harboring the respective codon-exchanges were amplified and cloned in the same manner. For RBD-RFP expression, RBD was amplified from the synthetic RBD DNA fragment with RBD-3F and RBD-5R, XbaI/BamHI digested and cloned into expression vector p48 (Hüttner et al., 2014b).