Conditioned freestyle 293 expression medium

Wild-type FB cDNA sequence (accession number NM_001710) additionally containing six histidine codons at 3′ terminus, as well as sequences for D279G, F286L, K323E, Y363A variants, and the quadruple mutant containing all aforementioned substitutions were codon optimized, synthesized and cloned into pCEP4 vector in the framework of GeneArt Gene Synthesis service by Thermo Fisher. Proteins were expressed and purified as described [23 (link)]. Briefly, vector DNA was transfected into HEK293 Freestyle cells using Freestyle Max reagent (Thermo Fisher). Conditioned FreeStyle 293 expression medium (Thermo Fisher) was collected at days 2, 4 and 7 post-transfection and stored at − 80 °C until the protein purification. The resulting proteins were purified with HisTrap FF affinity column (GE Healthcare) and elution was carried out with an imidazole gradient.

Wild-type C2 cDNA sequence (accession number NM_000063.5) additionally containing six histidine codons at 3′ terminus, as well as sequences for single, double, triple and quadruple variants, were codon-optimized, synthesized and cloned into pCEP4 vector in the framework of GeneArt Gene Synthesis service by Thermo Fisher (Waltham, MA, USA) as described in [16 (link)]. Plasmid DNA was transfected into HEK293 Freestyle cells (Thermo Fisher) using Freestyle Max reagent (Thermo Fisher). Conditioned FreeStyle 293 expression medium (Thermo Fisher) was collected at the 2nd, 4th and 7th days post-transfection and stored at −80 °C until the protein purification. The resulting proteins were purified with HisTrap FF affinity column (GE Healthcare) (Chicago, IL, USA), and elution was carried out with an 0.7 M imidazole gradient. Buffer was exchanged to PBS and the purity was confirmed by SDS-PAGE followed by Coomassie staining.