Protein expression in E. coli and purification

Recombinant proteins with polyhistidine tags were expressed in JM109(DE3) bacterial strain at 20°C, induced with 100 μM IPTG, then harvested and resuspended in TN buffer (50 mM Tris/HCl buffer pH7.5, 300 mM NaCl). cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO) (1 tablet/10 ml), DNAse (25 U/ml, Sigma-Aldrich), MgCl₂ (1 mM) and lysozyme (1 mg/ml, Sigma-Aldrich) were also added. The resuspension was frozen overnight at −20 °C. The following day it was thawed at room temperature, centrifuged at 40,000 × g at 5 °C for 10 min to remove cell debris, and purified by nickel affinity chromatography (Qiagen, Hilden, Germany). For Ca²⁺ sensors, proteins were further purified by size exclusion chromatography with Superdex 200 (GE Healthcare, Chicago, IL). Buffer composition for the size exclusion chromatography was 10 mM HEPES (pH 7.4), 150 mM NaCl and 100 µM EDTA. The buffer was exchanged with desalting column (PD-10, GE Healthcare) to HEPES buffer (10 mM, pH 7.4) supplemented with 150 mM NaCl, for all proteins unless otherwise noted.