Freestyle 293 f system

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.

Antibodies and Fab were expressed in CHO cells (44 (link)). Capture from cell culture media was accomplished by affinity chromatography using either MabSelect SuRE (Cytiva Life Sciences) for antibodies or CaptureSelect KappaXL (Thermo Fisher Scientific) for Fab. Subsequent purification was done by size exclusion chromatography using Superdex 200 for antibodies and Superdex 75 for Fab (Cytiva Life Sciences).
Human CD19 ECD (amino acids 21–289) was fused to the N-terminus of C-terminally His-tagged human serum albumin (HSA) and expressed in the FreeStyle 293-F system (Thermo Fisher Scientific). Protein was captured from cell culture media by HisPur Ni-NTA (Thermo Fisher Scientific) and further purified by Superdex 200 (Cytiva Life Sciences). Purified CD19-HSA-His was biotinylated with EZ-Link Sulfo-NHS-LC biotin (Thermo Fisher Scientific).