Expression plasmid for human TMEM2ECD 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 TMEM2ECD, 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. TMEM2ECD 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 TMEM2ECD proteins were eluted with PBS containing 0.5 M imidazole and 10% glycerol. TMEM2ECD proteins were further purified by size-exclusion chromatography on HiLoad 26/600 Superdex 200 (Cytiva, 28989336). Concentration of purified TMEM2ECD proteins was determined by measuring absorbance at 280 nm.
Hiload 26 600 superdex 200
Manufactured by Cytiva
The HiLoad 26/600 Superdex 200 is a size exclusion chromatography column designed for preparative-scale protein purification. It has a bed volume of 320 mL and is suitable for separating biomolecules with molecular weights ranging from 10,000 to 600,000 Da.
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2 protocols using hiload 26 600 superdex 200
1
Producing Recombinant TMEM2 Ectodomain
2
SARS-CoV-2 RBD Protein Production
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The expression plasmid encoding for codon-optimized SARS-CoV-2 RBD (329–538) with a C-terminal hexa-histidine tag was transfected into Expi293™ GnTI- (Thermo Fisher) cells (1×106 cells/mL) with PEI-Max. One week post-transfection, the clarified supernatant was purified on Ni-NTA column (Cytiva), followed by size-exclusion chromatography on HiLoad 26/600 Superdex 200 (Cytiva) equilibrated with PBS buffered saline pH 7.4.
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