Recombinant FMOD Protein Production

cDNA of a human FMOD transcript (Genbank assessor number: NM_002023) was subcloned into a commercially available vector pSecTag2A (Life Technology, Grand Island, NY) with C-terminal His-tag, and transfected into CHO-K1 cells (ATCC, Manassas, VA)^{19 (link)}. After establishing a stable expression clone, the FMOD was produced and purified by a contract research organization, GenScript (Piscataway, NJ). Briefly, a stable human recombinant FMOD-expressing CHO-K1 cell line was cultured in 1L serum-free Freestyle CHO Expression Medium (Thermo Fisher Scientific, Canoga Park, CA) at 37°C with 5% CO₂ in an Erlenmeyer flask. Cell culture supernatant was harvested on day 10 for purification with HiTrap™ IMAC HP, 1-mL column (GE Healthcare, Uppsala, Sweden). The fractions from a 100 mM imidazole elution were collected and dialyzed against 20 mM phosphate-buffered saline (PBS), pH 7.4. After that, the sample with low conductivity was loaded onto HiTrap™Q HP 1-mL column (GE Healthcare) for further purification. FMOD was then purified under non-reducing conditions, dialyzed again^{25 (link)}, and then subjected to lyophilization. The purity of the FMOD product is 85%. FMOD is reconstituted in PBS, followed by sterilization through a 0.22-μm filter (Thermo Fisher Scientific) before usage.