10 k centrifugal filter

Overall expression of the EFhd2 core domain was similar to that of EFhd1 ΔNTD. Cells transformed with the EFhd2 core domain were harvested by centrifugation, and the cell pellet was suspended in a lysis buffer [50 mM HEPES-NaOH (pH 7.5), 300 mM NaCl, 0.4 mM PMSF and 14.3 mM β-ME], lysed by sonication and centrifuged at 14 000g for 50 min at 4°C. The supernatant was then subjected to GST-bind agarose (Elpis) affinity chromatography. After washing with the lysis buffer, the target protein was eluted with lysis buffer supplemented with 30 mM gluta­thione, and the eluted protein was incubated with TEV protease overnight at 4°C to cleave the N-terminal GST-TEV tag. The target protein was further purified through a HiLoad 16/60 Superdex 75 gel-filtration column (GE Healthcare Life Sciences) pre-equilibrated with the final buffer [20 mM HEPES-NaOH (pH 7.5), 150 mM NaCl]. To obtain Zn²⁺-bound EFhd2 protein, the purified protein was treated with a 15-fold excess of EGTA and EDTA for 30 min at 4°C to remove pre-bound metal ions. The protein was then dialyzed in the final buffer for 24 h at 4°C, changing the buffer every 8 h. The dialyzed protein was concentrated using a 10 K centrifugal filter (Millipore) to 9.4 mg ml⁻¹ and treated with 0.75 mM ZnCl₂. The resultant Zn²⁺-bound protein was stored at −80°C.

To investigate their actin-binding and bundling activities, we purified full-length EFhd1 and EFhd2. The protein expression and purification of EFhd1 and EFhd2 were performed as previously reported (Mun et al., 2021 ▸ ). The two proteins were finally purified through a HiLoad 16/60 Superdex 75 gel-filtration column (GE Healthcare Life Sciences) pre-equilibrated with the final buffer containing 20 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 0.8 mM PMSF and 5 mM DTT. The purified protein was then concentrated using a 10 K centrifugal filter (Millipore) and stored at −80°C.
During purification, the presence of full-length EFhd1 and EFhd2 proteins was confirmed using SDS–PAGE.