Ni sepharose high performance resin

The cDNA encoding the Venus fluorescent protein fused to an N-terminal His-tag was inserted into the pET15b vector using Gibson Assembly (New England Biolabs). E. coli BL-21 (DE3) cells, transformed with the expression plasmid, were cultivated in LB medium with 100 μg/mL ampicillin at 37°C until they reached an OD₆₀₀ of approximately 0.55. Induction was carried out with 0.5 mM IPTG, followed by incubation at 37°C for 4 h under shaking conditions. Cells were harvested via centrifugation at 9,100 × g for 10 min at 4°C and subsequently resuspended in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5 mM imidazole, ×1 protease inhibitor (Nakalai tesque), 10% glycerol, and 5 mM 2-mercaptoethanol. Cell disruption was conducted via sonication using a sonicator (TOMY, UD-211). Ni Sepharose High Performance resin (Cytiva) was added to the bacterial extract supernatant and mixed using rotation at 4°C for 1 h. The slurry was loaded onto an empty column and washed thrice with lysis buffer. Protein elution was conducted using an elution buffer composed of 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10% glycerol, 500 mM imidazole, and 5 mM 2-mercaptoethanol. The purified His-Venus protein was then buffer-exchanged into PBS phosphate-buffered saline using a PD-10 column (Cytiva), portioned into aliquots, and stored at −80°C.

Transcription and translation for the production of biotinylated or FLAG-tagged recombinant proteins were performed using the WEPRO 7240 Expression Kit (Cell Free Science) as reported previously (Tanigawa et al, 2019 (link)). For purification of RhoB-GTP, His-RhoB (Q63L) recombinant protein was produced using the WEPRO7240H Expression Kit (Cell Free Science). The crude proteins were incubated with Ni Sepharose High Performance resin (Cytiva), and His-RhoB-GTP was eluted with elution buffer (20 mM phosphate, 300 mM NaCl, and 500 mM imidazole, pH 7.5). The eluted proteins were dialyzed in suitable buffers.