Endotoxin removal column

scaA, scaB, scaC, scaE, and tsa56 were amplified from the genomic DNA of O. tsutsugamushi Boryong strain by PCR using the primer pairs listed in S1 Table. The PCR products were cloned into pET-28a or pGEX4T-1 vector (Novagen, Gibbstown, NJ). Full length scaA genes were also amplified from the genomes of Boryong, Gilliam, Karp, and Kato strains for sequence comparison. All constructs were sequenced to confirm in-frame cloning. Recombinant Sca and TSA56 proteins were purified from E.coli BL21 (DE3) harboring a recombinant plasmid encoding each bacterial protein. Following induction with isopropyl β-D-thiogalactoside (IPTG) (0.1 mM, Duchefa, Zwijndrecht, Netherlands) at 16°C for 16 h, the proteins were purified using Ni-nitrilotriacetic acid His-resin (Qiagen, Calrsbad, CA) or glutathione-Sepharose 4B columns (GE Healthcare, Piscataway, NJ) according to manufacturer’s instructions. The purified proteins were dialyzed against phosphate-buffered saline (PBS) in an Aside-A-Lyzer Dialysis Cassette (Therrmo scientific, Rockford, IL) at 4°C for overnight. After dialysis, purified proteins were treated with endotoxin removal columns (Thermo scientific) and endotoxin contamination was determined using the QCL-1000 kit (Lonza, Bloemfontein, South Africa) according to manufacturer’s instructions. All protein contained less than < 0.05 EU/mg of endotoxin.

Transformed E. coli BL21(DE3) cells were grown in Overnight Express autoinducing medium (Formedium) containing 100 μg/mL ampicillin for 16 h at 37 °C. Cells were harvested by centrifugation at 4000× g for 20 min and lysed using B-PER lysis reagent (Thermo Fisher Scientific, Frederick, MD, USA)). Soluble material was clarified by centrifugation of the lysate at 12,500× g for 20 min at 4 °C, followed by 0.22 μm filtration. The filtrate was applied to Q-Sepharose (Sigma Aldrich Ltd., Dublin, Ireland)) resin equilibrated with 50 mM Tris-HCl pH 7.0 as a negative ion-exchange step to remove E. coli proteins. The unbound fraction was applied to Chelating Sepharose Fast Flow resin (GE Healthcare Bio-Science AB, Uppsala, Sweden) pre-charged with 50 mM NiSO₄ and equilibrated with 150 mM NaCl and 50 mM Tris-HCl, pH 7.9. Following five column volume washes with this buffer containing 20 mM imidazole, proteins were eluted with 0.5 M imidazole and buffer was exchanged into either PBS or DMEM for further analysis. Prior to cell infection experiments, endotoxins were removed using Endotoxin removal columns (Thermo Fisher Scientific, Frederick, MD, USA) according to the manufacturer’s instructions.

Protein expression vector pET28a-mCherry-CNA35 was obtained from Addgene (#61607). Transformed Escherichia coli (BL21) was cultured in 400 ml LB, and induced protein expression with isopropyl β-D-1-tthiogalactopyranoside for 20 hr at 25℃. Cultured bacteria were spun down, and collected bacteria pellet was sonicated. The lysate was centrifuged, and the resulting supernatant was run through a column filled with N-NTA His-band resin (Millipore). Bound protein was eluted and then dialyzed for overnight in PBS at 4℃. Endotoxin was removed by endotoxin removal columns (88274; Thermo) following with manufacturer’s protocol.

LPS-deficient Salmonella serovar Typhimurium X4700, provided by Dr. R. Curtiss (Arizona State University, Tempe, AZ), was used to purify flagellin using a modified acid-shock protocol. Overnight LB broth cultures were centrifuged, washed, and resuspended in PBS before acid treatment to liberate flagellin. Monomeric flagellin was prepared by depolymerizing dialyzed samples at 70°C for 1 h and passed through endotoxin removal columns (Pierce Biotechnology, Rockford, IL). Flagellin purity was determined using silver-stained SDS gels and endotoxin detection kits (Sigma, St. Louis, MO). Our flagellin preparations have previously been shown to have identical biological activity to recombinant flagellin produced in eukaryotic cells [10 (link)].

Recombinant GM-CSF, IL-4, and naïve CD4 T cell enrichment kit, Abs to IL-12p40/70, TNF-α, IL-6, IFN-γ, IL-17, PE-Cy7-CD11c, FITC-CD80, PE-CD86, efluor-MHCII, APC-CD40, STAT-1, pSTAT-1, STAT-4, pSTAT-4, JNK, and pJNK were purchased from BD Biosciences (San Diego, CA, USA). Biotinylated anti-CD83 and APC-conjugated CCR7 Abs were purchased from eBioscience (San Diego, CA, USA). Abs against actin were from Sigma (St. Louis, MO, USA), SOCS-3 from Abcam (Cambridge, MA, USA) and anti-pERK and ERK Abs from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The endotoxin removal column was purchased from Thermo Scientific (Rockford, IL, USA).