E coli bl21

The GST alone and GST fusion protein were expressed in E. coli BL21 (Takara), and purified by Pierce GST Spin Purification Kit (Thermo scientific). GST pull-down assay was performed using a Pierce GST Protein Interaction Pull-Down Kit (Thermo scientific). The purified GST-tagged fusion protein (BAIT) was immobilized on the Pierce Spin Column. MCF-7 cells were lysed in pull-down lysis buffer containing DNase (Takara). The supernatant was loaded onto the Pierce Spin Column, and then incubated at 4°C for 2 hour with gentle agitation. The column was centrifuged at 1250 × g for 1 minute and the flow through was discarded. Then the column was washed five times using wash solution. Elution buffer was added to the column followed by 5-min incubation with gentle agitation. After that, the column was centrifuged at 1250 × g for 1 minute, and the eluent was subjected to Western blot assay.

Synthetic recombinant plasmids were introduced into E. coli BL21 (Takara Bio). Transformed BL21 cells were cultivated with a small amount of 2×YT broth (containing 50 μg/mL ampicillin) in a shaker at 200 rpm and 37 °C and then inoculated into a large bottle of 2×YT at a dilution of 1:100. E. coli was grown at 160 rpm and 19 °C when the optical density at 600 nm (OD₆₀₀) reached 0.4–0.5. Then, 300 μM isopropyl-β-d-thiogalactopyranoside (Nacalai Tesque) was added to induce protein synthesis with overnight incubation at 160 rpm and 19 °C. Following centrifugation, bacteria were collected and suspended in lysis buffer (1 M NaCl, 50 mM Tris pH 8.0, 1% Triton X) containing protease inhibitor cocktails (Merck KGaA, Darmstadt, Germany) and sonicated. After centrifugation at 15,000 rpm for 10 min, the supernatants were collected and filtered through a 0.45 μm syringe filter, and the proteins were purified on a GSTrap FF column using an AKTA start liquid chromatography system (Cytiva, Tokyo, Japan) following standard protocols. Eluted proteins were fractionized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained for visualization with Coomassie brilliant blue G-250 (Bio-Safe CBB G-250 Stain, Nacalai Tesque).