Expression of Full Length PRCV ISU-1 Spike Protein

The spike sequence from PRCV ISU-1 (GenBank Accession number: {"type":"entrez-nucleotide","attrs":{"text":"DQ811787.1","term_id":"110746812","term_text":"DQ811787.1"}}DQ811787.1) was modified to replace the coding region for the C-terminal transmembrane and cytoplasmic domains, with a T4 foldon trimerization domain followed by a His⁸ tag. The sequence coding for amino acids E914 and L915 was mutated to two prolines in an attempt to stabilise the spike in a pre-fusion state similar to that described for other coronavirus spikes. The sequence was optimised to human codons and synthesised into the plasmid expression vector pTwist CMV BetaGlobin, to create vector pPRCV-ISU1-PP-Foldon. The spike was expressed in Expi293F cells (ThermoFisher) according to the manufacturer’s instructions. Culture supernatants were clarified by centrifugation and purified through 5 mL HisTrap FF column (GE Healthcare). Fractions containing spike were concentrated and the excess imidazole removed by using buffer exchange columns (Zeba, Merck).