Producing Recombinant TMEM2 Ectodomain

Expression plasmid for human TMEM2^ECD fused with a C-terminal 6× His tag was constructed by inserting the cDNA into pSecTag2B, in which the original Myc/His tag was inactivated by inserting a stop codon. For the production of mouse TMEM2^ECD, the construct used for the expression of a tag-less TMEM2 ectodomain (11 (link)) was modified by inserting a 6× His tag at its C terminus. TMEM2^ECD proteins were produced using the FreeStyle 293-F system (ThermoFisher, R79007), as described previously (42 (link)). Culture supernatants were first applied to HisPur Cobalt Resin (ThermoFisher, R79007), and bound TMEM2^ECD proteins were eluted with PBS containing 0.5 M imidazole and 10% glycerol. TMEM2^ECD proteins were further purified by size-exclusion chromatography on HiLoad 26/600 Superdex 200 (Cytiva, 28989336). Concentration of purified TMEM2^ECD proteins was determined by measuring absorbance at 280 nm.