Purification of His-tagged Protein from E. coli

A single colony of E. coli harboring pRham N-His Kan::LysKB317 (Table 1) was isolated and inoculated into 5 mL LB media with Kan at 37°C and 200 rpm shaking overnight. This was used to inoculate fresh 25 mL LB with Kan at a ratio of 1:100, which was then incubated at 37°C and 200 rpm until the OD_600 nm reached 0.6 (Lu et al., 2020 (link)). Culture was induced with 0.2% (w/v) L-rhamnose (Sigma-Aldrich Inc.) for at least 16–18 h. Cells were centrifuged at 8,000 x g for 15 min at 4°C, and the spent medium was decanted. Cell pellets were then lysed using B-Per (Invitrogen) with DNase I, RNase I, and lysozyme (10 U/mL; ThermoFisher) and incubated at room temperature for 20 min. The whole cell lysate was centrifuged at 15,000 x g for 5 min and the soluble supernatant was pipetted into a His-Spin column (ZymoResearch) following the manufacturer’s protocol for Ni-NTA protein purification. The concentration of purified protein was quantified using the Qubit Protein Assay Kit (ThermoFisher) and subsequently used as a positive control for SDS-PAGE and Western blot analysis.