Isopropyl β d thiogalactoside

The tsa56 gene was amplified from the genomic DNA of O. tsutsugamushi Boryong genotype by PCR and a recombinant gene encoding cTSA56 (concatenated CBs of TSA56 Boryong) was synthesized after codon optimization (Bionics, Seoul, South Korea). Primers and sequence information of the recombinant proteins are available in Supplementary Table S3. The genes were cloned into pET-28a (Novagen, Gibbstown, NJ, USA) via EcoRI and SalI sites. TSA56 and cTSA56 proteins were purified from E.coli BL21 (DE3) harbouring a recombinant plasmid encoding each protein. Following induction with isopropyl β-D-thiogalactoside (0.1 mM, Duchefa, Zwijndrecht, Netherlands) at 16°C for 18 h, the proteins were purified using Ni-nitrilotriacetic acid His-resin (Qiagen, Calrsbad, CA, USA) according to the manufacturer's instructions. After dialysis against phosphate-buffered saline (PBS), purified proteins were treated with endotoxin removal column (Thermo scientific) and endotoxin contamination was determined using the QCL-1000 kit (Lonza, Bloemfontein, South Africa) according to the manufacturer's instructions. All protein contained less than <0.05 EU/mg of endotoxin. The identity and purity (>90%) of proteins were assessed by Western blotting and Coomassie blue staining, respectively.

Recombinant p24 proteins were purified from E. coli as previously described (59 (link)) with minor modification. For the expression and purification of the fusion protein, E. coli BL21 strains (RBC Bioscience, Taipei City, Taiwan) were transformed with pET23a-p24. Protein expression was induced by adding 0.4 mM isopropyl β-d-thiogalactoside (Duchefa Biochemie, Haarlem, Netherlands). Bacterial cells were harvested and disrupted by sonication on ice for 10 min. Sonicated lysates were centrifuged at 1,600 × g for 20 min at 4°C, and the pellets containing p24 protein were resuspended in binding buffer containing 4 M urea (Sigma Aldrich, St. Louis, MO, USA). The proteins were purified using Ni-NTA His binding resin (Merck, Darmstadt, Germany) and eluted with elution buffer (300 mM NaCl, 50 mM sodium phosphate buffer, 250 mM imidazole) containing 4 M urea. Purified proteins were dialyzed serially against the elution buffer to remove imidazole, urea, and residual salts. Purity of p24 protein was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12% gel). The gel was visualized using Coomassie brilliant blue staining methods (60 (link)) (Figure S9 in Supplementary Material).

A cell-permeable Tat expression vector was prepared as described in a previous study [27 (link)]. Sense primer 5′-CTCGAGATGCGCCTCCGC-3′ and antisense primer 5′-GGATCCTTAGAGATCCTCCTGTGCC-3′ were used to amplify cDNA for CRIP1a by PCR. The PCR product was subcloned in a TA cloning vector (pGEM^®-T easy vector; Promega Corporation, Madison, WI, USA) and ligated into the Tat expression vector to produce a Tat-His-CRIP1a fusion protein. The Tat domain consists of 9 amino acids, RKKRRQRRR, and is connected with a 6xHistidine tag. They are inserted in the N-terminal of CRIP1a. A His-CRIP1a (Control-CRIP1a) without the Tat domain was also prepared to use as a control.
The Tat-His-CRIP1a and His-CRIP1a-containing plasmids were transformed into Escherichia coli BL21 cells to produce both proteins. Isopropyl-β-d-thiogalactoside (0.1 mM, Duchefa, Haarlem, The Netherlands) was given to the bacterial cells at 18 °C for 8 h, and the harvested cells were purified with a Ni²⁺-nitrilotriacetic acid Sepharose affinity column and PD-10 column chromatography (Amersham, Braunschweig, Germany). To estimate the concentration of purified proteins, a Bradford assay was performed.