Ni nta resin column

Sodium hydroxide (NaOH, 98%) and endotoxin removal spin columns were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Tetrachloroauric(III) acid trihydrate (HAuCl₄•3H₂O, 99%) was purchased from Alfa Aesar (Haverhill, MA, USA). Tryptone, yeast extract, sodium chloride (NaCl), kanamycin, isopropyl β-D-1-thiogalactopyranoside (IPTG), DNase I, lysozyme, and ampicillin were purchased from BioShop (Burlington, ON, Canada). The proteinase inhibitor and Ni-NTA resin column were purchased from Sigma-Aldrich (St. Louis, MO, USA). Coomassie R-250 dye was purchased from AMRESCO (Solon, OH, USA). Amicon^® Ultra centrifugal filters (3K MWCO) were purchased from Merck Millipore (Burlington, MA, USA).

Bacterial pellets were re-suspended in 100 mL of lysis buffer (one tablet of proteinase inhibitor, 2 μg/mL DNase I, and 50 mg/mL lysozyme) under sonication at 4 °C. After sonication, lysates were centrifuged at 12,000× g and 4 °C for 30 min. Supernatants were filtered (through a pore size of 0.45 μm), and an Ni-NTA resin column (Sigma-Aldrich) was further used to purify the derived Hp peptide. Before purification, the Ni-NTA resin column was equilibrated with 10 mM of imidazole/PBS. After bonding to the Ni-NTA resin, the derived Hp peptide was eluted by a linear gradient of a 20 to 500 mM imidazole/PBS program. The eluted peptide was directly analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie R-250 dye, as shown in the Supporting Information (Figure S2). The 3D structure of the derived Hp peptide is shown as Figure S3 in the Supporting Information. The derived Hp peptide was concentrated with Amicon^® Ultra-centrifugal filters (3K MWCO) and then dialyzed with PBS (at pH 8.0). After dialysis, the derived Hp peptide was immediately processed with endotoxin-removal spin columns and sterile filtration (0.22 μm) and stored at 4 °C for subsequent experiments.