To express recombinant ChpBK protein [National Center for Biotechnology Information (NCBI reference sequence: NP_418646.1], E. coli BL21(DE3) strain carrying PET-28a-chpBK plasmid was grown in lysogeny broth (LB) medium (Sangon Biotech, A507002) supplemented with Kan (10 μg/ml) at 37°C and 200 rpm until the optical density at 600 nm (OD600) reach to 0.5. Isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM, and cell growth resumed for 2 hours under the same conditions. Harvested cell pellets were lysed by sonication in 30 ml of buffer A [10 mM bis-tris (pH 7.0) and 30 mM imidazole] and centrifuged for 20 min at 16,000g and 4°C. The soluble fraction was incubated on ice for 30 min in the presence of DNase and RNase (10 g/ml; Thermo Fisher Scientific). The His-ChpBK fusion protein was purified from clarified lysates using Ni-NTA (nickel-nitrilotriacetic acid) beads (Invitrogen). Protein purity was determined by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and samples were quantified using the Bio-Rad protein assay and stored at −80°C.

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