Ni2 chelating sepharose column

To obtain soluble proteins, we expressed recombinant ACP proteins using the pET32a system, a thioredoxin (TRX) fusion system containing a 6×His tag to facilitate the purification on a Ni²-chelating Sepharose column and a partner TRX to help the proteins fold correctly. The ClonExpress^®II One Step Cloning Kit (Vazyme) was used to insert the coding regions of mature bfACP3 (Asp24 to Ala561) and bfACP5(Asp21 to Glu636) into the pET32a plasmids. The plasmids were transformed into E. coli BL21 (DE3). The transformed bacteria were cultured to an OD=0.6-0.8 and induced with 1 mM IPTG at 37°C for four hours. After induction, the bacterial cells were collected and sonicated for lysis. The cell lysis supernatant was purified through a Ni²-chelating Sepharose column (GE Healthcare). The recombinant proteins were eluted with 250 mM imidazole, dialyzed in PBS buffer at 4°C for 12 hours three times and concentrated by ultrafiltration using an Ultrafree centrifugal filter device (Millipore). Pierce™ BCA Protein Assay Kit (Thermo) was used to determine the protein concentration according to the manufacturer’s protocol. Primers used for preparation of recombinant proteins were listed in Table 1.

Recombinant His-tagged S100A9 and galectin-1 were prepared as described previously [16 (link)]. Target genes were cloned into pET28a vector (Novagene, Bedford, MA, USA) and expressed in Escherichia coli strain BL21 after isopropyl b-D-1-thiogalactopyranoside induction and purified by a Ni²⁺-chelating Sepharose column (GE Healthcare, Chicago, IL, USA). The endotoxin was removed by dialysis against buffer with 1% Triton X-114 before use. S100A8 (ProSpec, Rehovot, Israel) and S100A8/A9 (BioLegend, San Diego, CA, USA) were purchased from commercial sources.

H. pylori genomic DNA was extracted using DNA purification kits (Qiagen, Venlo, Netherlands) and the genes encoding AlpA, OipA, BabA, and SabA were cloned using the expression vector pET28a (Novagene, Bedford, MA); the sequences of the primers used are listed in Supplemental Table V. Expression of each protein, bearing a 6-His tag, was induced in E. coli strain BL21 by incubation for 4 h at 37 °C with 1 mM isopropyl β-D-1-thiogalactopyranoside (Sigma, St. Louis, MO), then the recombinant proteins were dissolved in binding buffer (20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, pH 7.9, containing 8 M urea) and purified on a Ni²⁺-chelating Sepharose column (GE Healthcare) by elution with binding buffer containing 200 mM imidazole. All proteins were >95% pure, as shown by Coomassie Blue staining of SDS-PAGE gels.