Glutathione sepharose 4b 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.

The full-length of scaB gene (nucleotide 1–1,950) was amplified from the genomic DNA of the O. tsutsugamushi Boryong and Kuroki genotype by using the primer pairs listed in Table 1. The PCR product from Kuroki genotype is directly cloned into pCR™2.1 vector for sequencing. To generate the recombinant ScaB protein, a gene fragment corresponding to the ScaB passenger domain (amino acids 23–372) was amplified from the Boryong genomic DNA by using the primer pair listed in Table 1. The amplified fragment was then directionally cloned into pET28a or pGEX4T-1 vector (Novagen, Gibbstown, NJ, United States).
For the expression and purification of ScaB proteins, E. coli BL21 (DE3; Novagen) was transformed with ScaB passenger domain then 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, Carlsbad, CA, United States) or glutathione-Sepharose 4Bcolumns (GE Healthcare, Piscataway, NJ, United States) according to manufacturer’s instructions. The purified proteins were dialyzed against phosphate-buffered saline (PBS) in a Slide-A-Lyzer Dialysis Cassette (Thermo scientific, Rockford, IL, United States) at 4°C for overnight.

cDNA encoding candidate proteins identified by liquid chromatography–mass spectrometry (LC–MS) analysis (described elsewhere [8 (link)]) were cloned from a murine brain cDNA library. cDNA constructs were inserted into the pGEX6P-3 plasmid (GE Healthcare, USA) and expressed as GST-fusion proteins in High-efficiency BL21(DE3) Competent Cell (GMbiolab, Taiwan). GST-fusion proteins were purified using glutathione-Sepharose 4B columns (GE Healthcare, USA). Protein-bound glutathione beads were thoroughly washed with cold PBS 6 times, and thereafter eluted with 20 mM reduced glutathione (recombinant GST or GST-fusion peptides) or incubated with PreScission Protease (GE Healthcare, USA) overnight at 4°C to remove the GST tag. These recombinant proteins were incubated with Affi-Prep Polymyxin Media (Bio-Rad, USA) for 4 h at 4°C to remove endotoxins and endotoxin-bound proteins. To generate modified DJ-1 proteins, which lack the ability to form its oxidized state, recombinant DJ-1 protein was treated with 1 mM dithiothreitol and incubated with 55 mM iodoacetamide (Wako Pure Chemical Industries, Japan) in a dark chamber at room temperature for 1 h. We examined the purity of these recombinant proteins by SDS-PAGE with Coomassie brilliant blue (CBB) staining. Recombinant GST protein was used as a negative control for cytokine induction in BMMs.

GST-MITF fusion protein was purified from E. coli and immobilized on glutathione Sepharose 4B columns (GE Healthcare). HEK293T cells transfected with the indicated plasmids were lysed in NETN buffer with protease inhibitor cocktail (Sigma-Aldrich, USA) and incubated with Sepharose beads immobilized with the indicated GST-tagged proteins at 4 ℃ overnight. After washing the beads were boiled in 60 µL 2×SDS loading buffer and subjected to immunoblotting with the indicated antibodies.

For testing the direct interaction of NdhM with Ndh subunits, the fragments containing ndhH, ndhI, ndhJ, ndhK, ndhN, ndhO, and ndhS genes were amplified by PCR and cloned into pET28a, respectively, to form His-tagged fusion protein constructs. The fragment containing ndhM was cloned into pGEX-4T-1 to form the GST-tagged fusion construct. Primer sequences used are listed in supplemental Table 1. These constructs were transformed into E. coli strain BL21(DE3) pLysS and induced by 1 mm isopropyl β-d-thiogalactoside for 16 h at 16 °C. These fusion proteins were purified using a nickel column (GE Healthcare) and glutathione-Sepharose 4B column (GE Healthcare), respectively, according to the manufacturer's instructions.