Ni affinity chromatography column

E. coli DH5α cells were used for general DNA recombinant techniques and BL21 (DE3) cells were used for MDH expression. DH5α and BL21 (DE3) competent cells were purchased from Real Biotech Corporation (Taipei, Taiwan). Cells were grown in 2YT medium (16 g yeast extract, 10 g tryptone, and 5 g NaCl per litre) supplemented with appropriate antibiotics. Restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA). DNA polymerase and ligase were from Takara (Shiga, Japan). Ni-affinity chromatography column was from GE Healthcare (Little Chalfont, UK). All chemicals except formaldehyde used in enzymatic reactions, including the cofactors NAD⁺ and NADH, were purchased from Sigma-Aldrich (St. Louis, MO, USA). And methanol-free 16% formaldehyde was from Thermo Scientific (Waltham, MA, USA).

The recombinant plasmid pET28a-hupB, which was C-terminally tagged with 6 × His, was electronically transformed into the host strain BL21(DE3). The positive strain was cultured at 37°C in LB broth with a final concentration of 50 μg/ml kanamycin. After about 3 h of incubation, the OD₆₀₀ reached about 0.5, and 0.1 mM Isopropyl β- d-1-thiogalactopyranoside (IPTG) was added to the culture solution to induce HupB expression at 16°C for about 12 h. After washing with PBS, the culture was resuspended with a binding buffer for ultrasound, which was composed of 50 mM Tris-HCl, 300 mM NaCl, and 10 mM imidazole. The fusion protein was then purified from the soluble extracts using a Ni-affinity chromatography column (GE Healthcare, #10280275) (Rowe and O’Gara, 2016 (link)). The protein concentration was evaluated by the Bicinchoninic Acid Assay (Sangon Biotech, #C503021) and stored at −80°C for further use.

Positive colonies were confirmed by DNA sequencing. Expression and purification was conducted using transformed cells grown in LB medium (5 ml) with kanamycin (50 μg ml^-1) with shaking at 220 rpm at a temperature of 37°C for 12 h. Cultures were diluted to 1000 ml with LB medium and grown to an OD₆₀₀ value of 0.4–0.6. To induce protein expression, 0.1 mmol L^-1 isopropyl-beta D-thiogalactopyranoside (IPTG) was added and culturing continued for 4 h at 37°C.
The expressed protein was obtained in soluble form in the supernatant, and purification was accomplished using a Ni affinity chromatography column (GE Healthcare, Uppsala, Sweden). Bound protein was eluted by digestion with recombinant bovine enterokinase at 26°C for 15 h to remove the His-tag. Expression and purification were verified by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein concentration was measured as described by Cao et al. (2017) (link). Dialysis of purified protein was carried out in 30 mM Tris-HCl buffer [Tris(hydroxymethyl)aminomethane] at pH 7.4 and pH 5.0 before fluorescence binding assays, and protein was stored at -80°C until use.