Lemo21 de3 cells

The target amino-acid sequence (NCBI Reference Sequence WP_049406473) was back-translated and codon usage was optimized for expression in E. coli with OPTIMIZER (Puigbò et al., 2007 ▸ ). A plasmid encoding the SmTetX protein including a 6×His tag at the N-terminus of the translated protein was synthesized and cloned into the pET-28a(+)-TEV expression vector via NdeI and BamHI restriction sites by GenScript, USA. The cleavage site for Tobacco etch virus (TEV) protease was placed between the His tag and the target SmTetX sequence (Supplementary Fig. S1).
The plasmid was transformed into competent E. coli strain Lemo21 (DE3) cells (New England Biolabs) using the heat-shock method. Cell precultures in LB medium were incubated in a shaker at 32°C and 180 rev min⁻¹ overnight. The medium was supplemented with 50 µg ml⁻¹ kanamycin and 25 µg ml⁻¹ chloramphenicol. 10 ml preculture was added to 1 l Power Broth medium (Molecular Dimensions, catalogue No. MD12-106) and the cell culture was incubated at 30°C and 150 rev min⁻¹ until the OD₆₀₀ reached ∼0.5. Expression was induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 20°C for 16 h. The cells were harvested after centrifugation of the culture at 4°C and 5000g for 30 min.

The fragments of Stim1 (342–448, 344–382 and 408–442) and Orai1 (1–103, 1–47, 48–103, 228–301, 245–272 and 272–292) were amplified from full length constructs and cloned into pMAL-c5X (New England Biolabs) or pGEX-4T2 (Addgene) vector, in-frame with MBP or GST protein coding sequences, respectively. The fusion proteins were expressed in LEMO21(DE3) cells (New England Biolabs) by inducing with IPTG (0.3 mM) for 14 hours at 18°C. The MBP- or GST-tagged proteins were purified by immobilizing on amylose (NEB) or glutathione resin (Qiagen), respectively. The resin bound proteins were incubated with 20–50 nM of α-SNAP protein in Ringer’s buffer containing 0.1% NP-40 and 2 mM imidazole for 1 hr at 4°C. The resin was washed thrice with Ringer’s buffer, boiled in SDS sample buffer and subjected to SDS-PAGE followed by Western Blot analysis.

Enzyme variants were expressed in Lemo21(DE3) cells (New England Biolabs, Ipswich, MA, USA), and purification done in one step on a StrepTactin column by a protocol that has been described elsewhere 19. The enzyme was eluted in 25 mm Tris, pH 8.0, 10 mm MgCl₂, 2.5 mm desthiobiotin, with 15% v/v ethylene glycol added as a storage medium to prevent frost damage. Enzyme purity was judged to be 95–99% pure by SDS/PAGE on 4–12% bis‐Tris NUPAGE^® gels (Life technologies, Carlsbad, CA, USA). Enzymes were snap‐frozen and stored at −20 °C and never used in analysis after more than one freeze/thaw cycle. For denaturation and refolding studies, the enzyme was concentrated on HiTrap Q XL (GE Healthcare, Chicago, IL, USA) ion exchange column on a low‐pressure Bio‐Rad BioLogic LP Chromatography System and eluted with a NaCl gradient from 0.0 to 1.0 m and then further concentrated on a 2.0‐mL Amicon Ultra centrifugation filter with a 30k cut‐off (Millipore, Burlington, MA, USA). Concentrated enzyme samples were aliquoted to 10 μL single use portions and stored at −20 °C.