Isopropyl o d thiogalactopyranoside

The C1 domain and REM motif of RasGRP1/3 in the pGEX-2T and pGEX-5X1 plasmids were transformed into BL21 (DE3) One Shot chemically competent E. coli (Invitrogen). Transformants were grown in LB broth medium (K-D Medical) at 37°C until the optical density of the bacterial suspension reached 0.6–0.8. Expression of the GST fusion proteins was induced with 0.3 mM isopropyl O-D-thiogalactopyranoside (Thermo Fisher Scientific) for 4 h at 37°C or 6 h at room temperature (C1 domains and REM motifs, respectively). Bacterial cells were subjected to B-PER bacterial protein extraction reagent (Thermo Fisher Scientific) or lysis buffer (150 mM NaCl, 50 mM Tris buffer, pH 7.4). The expressed GST-tagged C1 and REM proteins were purified using a GST Spin Purification Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Purification efficiency was evaluated by SDS-PAGE analysis. Purified proteins were stored in 20% glycerol at −80°C.

The full-length RasGRP1/3 and the REM chimeras in the pMAL-c5x plasmid were transformed into BL21 (DE3) One Shot chemically competent E. coli (Invitrogen). Transformants were grown in LB broth medium (K-D Medical) at 37°C until the optical density of the bacterial suspension reached 0.5–0.6. Expression of the MBP fusion protein was induced with 0.3 mM isopropyl O-D-thiogalactopyranoside (Thermo Fisher Scientific) for 6 h at room temperature. The expressed MBP-tagged proteins were purified using the pMAL™ Protein Fusion and Purification System according to the manufacturer’s instructions. Purification efficiency was evaluated by SDS-PAGE analysis. Purified proteins were stored in 20% glycerol at −80°C.