Purification and Refolding of MCU72-189

The pET-28a MCU_72-189 vector and associated D147R mutant were from the previously described work.³³ (link) WT or D147R MCU_72-189 constructs were transformed in BL21 (DE3) codon plus E. coli. Transformed cells were grown in LB medium containing kanamycin (60 μg mL⁻¹) at 37°C to an OD 600 nm of ∼0.6–0.8, and 200 μM of IPTG was added to induce expression over a 16 h period. The harvested cells were purified under denaturing conditions using (Ni-NTA) resin (HisPur) according to the manufacturer instructions. The protein was refolded in 20 mM Tris, 150 mM NaCl and 1 mM DTT, pH 8.8 by dialysis. After overnight thrombin cleavage (∼1 U/mg protein), the protein was further purified by SEC through a Superdex 200 10/300 GL (Cytiva) connected to an AKTA pure FPLC system (Cytiva) at 10°C. This final purification step buffer was 20 mM Tris (pH 8.5), 150 mM NaCl, 1 mM DTT. The protein concentration of MCU_72-189 was estimated using an extinction coefficient (280 nm) of 0.3693 (mg/mL)⁻¹ cm⁻¹.