Human embryonic kidney 293 Freestyle (HEK293F) cells were transfected with mammalian-codon-optimised genes encoding 2P-stabilised SARS MERS and HKU1 S proteins containing a C-terminal T4 fibritin trimerization domain, an HRV3C cleavage site, an 8xHis-tag and a Twin-Strep-tag41. H3N2 Victoria 2011 hemagglutinin was also expressed in the HEK293F cells. The 200 ml cultures were harvested 6 days after transfection, filtered and purified by nickel-affinity chromatography and size exclusion chromatography using a SuperdexTM 16/600 75 pg column (GE Healthcare).