Isopropyl β d thiogalactopyranoside iptg

Deletion strains were complemented with a bpeT or bpeEF-oprC gene(s) originating from strain 1026b or Bp82 utilizing the mini-Tn7 system, which allows stable and site-specific single-copy insertions into the B. pseudomallei genome at three possible glmS-associated attTn7 sites (50 (link)). The respective mini-Tn7 elements, along with an empty-mini-Tn7 element used as a control, were transferred to the target B. pseudomallei strains either via conjugation from E. coli or by electroporation, and glmS-associated insertions were verified as previously described (50 (link), 51 (link)). Mini-Tn7 insertions at the glmS2-associated attTn7 site were routinely retained for further studies, unless noted otherwise. The inducible E. coli trp/lac operon hybrid P_tac promoter was used for regulated expression of the bpeEF-oprC genes. BpeEF-OprC expression was induced by addition of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG; Gold Biotechnology, St. Louis, MO) (28 (link)).

Poly-ethylene glycol (PEG) 3350 was purchased from Sigma Life Sciences, tri-ammonium citrate was purchased from Sigma Life Sciences, Ampicillin was purchased from GoldBio, dehydrated Luria-Bertani (LB) Broth was purchased from Fisher Scientific, DL-dithiothroitol (DTT) and isopropyl-β-d-thiogalactopyranoside (IPTG) were purchased from GoldBio. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was purchased from Fisher BioReagents. Imidazole was purchased from Acros Organics; tris(hydroxymethyl)aminomethane (Tris) was purchased from Fisher Scientific. Sodium chloride (NaCl) was purchased from Fisher Chemical, and bovine serum albumin (BSA) was purchased from Sigma Life Science.

The gene encoding human apolipoprotein A-I (hApoA-I) spanning residues D44 to L243 (Δ1−43) was cloned into the pET21a vector (Novagen) containing a thrombin-cleavable hexahistidine (6×His) tag at the C-terminus^{50 (link)}. Recombinant protein was produced in Escherichia coli BL21 (DE3) cells cultured in LB medium at 37 °C. When the OD₆₀₀ reached 0.6, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG, GoldBio) was added and cells were incubated for a further 5 h at 37 °C. After harvesting cells by centrifugation at 7700 g for 10 min, the pellet was resuspended in lysis buffer containing 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM PMSF, and 10 μL/mL of DNase I. After sonication on ice, cell debris was removed by centrifugation at 30,000 g for 1 h. The supernatant was loaded onto Ni-NTA agarose resin (GoldBio), washed thoroughly with buffer containing 20 mM Tris-HCl pH 8.0, 200 mM NaCl, and 30 mM imidazole, and elution of bound proteins was performed using a gradient of imidazole up to 500 mM. The 6×His tag was removed by thrombin cleavage overnight and the protein was further purified by HiTrap Q (Cytiva) anion exchange and Superdex 200 Increase 10/300 GL gel filtration chromatography steps. All purification steps were performed at 4 °C.

A plasmid DNA Mini-Prep kit and all restriction enzymes were purchased from New England Biolabs (Beverly, MA, USA) and Elpis Biotech (Daejeon, Korea). The polymerase chain reaction (PCR) kit and gel extraction product kit came from Real Biotech Corp (Taipei, Taiwan). All oligomers were commercially synthesized by Bioneer (Daejeon, Korea). General chemicals were product of Sigma Aldrich (St. Louis, MO, USA) in the best grade available. The scFv-Cys3 gene was cloned in the bacterial expression vector pET-28a(+). We used this pET-28a(+)-scFv-Cys3 construct to express a recombinant scFv-Cys3 in Escherichia coli. The expression vector and Rosetta (DE3) were purchased from Novagen (Madison, WI, USA). Isopropyl-β-d-thiogalactopyranoside (IPTG) was obtained from Gold Biotechnology (St. Louis, MO, USA). Protein samples were analyzed on 12% polyacrylamide gels and stained with Coomassie Brilliant Blue R250. The gold chip was procured from MiCo NanoBioSys. SPR was performed using a MiCo SPR nano-instrument from MiCo NanoBioSys, using the gold sensor chip.