Isopropyl β d thiogalactoside iptg

Amino acid auxotrophs for methionine (Met), isoleucine (Ile), lysine (Lys), and leucine (Leu) harboring the pTAC-luc plasmid were prepared following a previously reported protocol [16 (link)]. Briefly, a tac promoter was added into the pETDuet-1 vector (Novagen, San Diego, CA, USA) to replace the T7lac promoter and generate the pTAC. The luc component was amplified using the pGL-Basic vector (Promega, WI, USA) and the PCR products were cloned into the pTAC to produce pTAC-luc. Finally, the pTAC-luc plasmids were transferred into competent cells by electroporation using a Gene Pulser system (BioRad, CA). M9 minimal medium, Luria-Bertani (LB) broth, ampicillin, kanamycin, isopropyl-β-D-thiogalactoside (IPTG), cyanocobalamin (vitamin B₁₂), dimethyl sulfoxide (DMSO), glycerol, ethylene glycol (EG), polyethylene glycol (PEG), sucrose, trehalose, agarose (low gelling temperature), and amino acids (Met, Ile, Lys, and Leu) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents and chemicals used in this study were of analytical grade.

The OMP29 was expressed by pET28B/omp29 E. coli BL21 (DE3) following the manufacturer's instructions (New England Biolabs, Ipswich, MA, United States). A single colony of pET28B/omp29 E. coli BL21 (DE3) was inoculated into LB broth with kanamycin (50 μg/ml) (Sigma–Aldrich, St. Louis, MO, United States) and incubated under agitation at 120 rpm for 16 h at 37°C. Cells were transferred to fresh LB broths, and incubated under agitation at 250 rpm at 37°C until O.D. 600 nm ≈ 1 was reached. Expression of Aa OMP29^His was induced by adding 1 mM of isopropyl β-D-thiogalactoside (IPTG, Sigma–Aldrich, St. Louis, MO, United States) at 37°C and the recombinant protein was observed after 2 h in culture precipitate. Cells were then lysed [27 (link)] and Aa OMP29^His in cells lysate supernatant was purified by affinity chromatography using nickel chelating resin (Ni-NDA, Invitrogen).

Glyceraldehyde-3-phosphate dehydrogenase recombinant protein was obtained as described by Barbosa et al. (2006) (link). Briefly, we used the TOPO-pET-100-GAPDH construct in order to produce recombinant GAPDH. The recombinant protein synthesis started by the addition of isopropyl-β-D-thiogalactoside (IPTG) (Sigma-Aldrich) to a final concentration of 1 mM. BL21 cells expressing recombinant P. lutzii GAPDH were incubated with 1 μ/mL lysozyme and underwent subsequent sonication on ice. For the recombinant GAPDH with his-tag at the N-terminal end purification, we used the nickel-nitrilotriacetic acid resin (Ni-NTA) (Invitrogen, Carlsbad, CA, United States). For 1 mL of lysate, we used 250 μL of resin. The lysate was incubated with the resin on ice under gentle agitation for 2 h. Then the resin was washed 5 times with native wash buffer (50 mM Na₂HPO₄, 20 mM imidazol, pH 8.0) in order to eliminate contaminants. The recombinant GAPDH bound to the resin was eluted with native elution buffer (50 mM Na₂HPO₄, 250 mM imidazol, pH 6.0). The purity and size of the protein were assessed by a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining.

4-(4-(2,3-Dihydrobenzo[1,4]dioxin-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl)benzamide (D4476) was from Abcam (UK); Interleukine-6 (IL-6), chloroquine (CQ) and isopropyl-Β-D-thio-galactoside (IPTG) were from Sigma-Aldrich (Italy).

The proteins were expressed in the E. coli BL21 (DE3) strain (Novagen), which was initially cultured at 37 °C until OD600 reached about 0.6 to 1. Next, 0.4 mM isopropyl β-D-thiogalactoside (IPTG, Sigma) was added to induce protein expression at 16 °C overnight. For purification, the supernatant from cell lysate was first loaded onto a Ni-NTA column (Cytiva), followed by further separation using three columns HiLoad 16/600 Superdex 75 pg (Cytiva), RESOURCE Q 6 mL (Cytiva), and Superdex 200 Increase 10/300 GL (Cytiva), respectively. Finally, the purity of the proteins reached above 95%. The proteins were concentrated to 6–10 mg/mL for crystallization and other assays. All samples were flash-frozen in liquid nitrogen and stored at −80 °C.

The d-lactate standard, NADH, NADPH, and isopropyl-β-d-thiogalactoside (IPTG) were obtained from Sigma-Aldrich (St. Louis, MO). Oligonucleotides and gene synthesis were carried out by Sangon Biotech Co., Ltd. (Shanghai, China). All other chemicals and reagents were of at least analytical grade and were available commercially.

The nucleotide sequence corresponding to the extracellular domain of CD38A (Figure S1) was synthetized and subcloned into the E3 expression vector (Abyntek) together with an N-terminal 6 x histidine tag. The recombinant plasmid was transformed into BL21 cells and a kanamycin-resistant single positive colony was then incubated at 37°C in Luria-Bertani (LB) media. When the OD600 reached 0.6, 0.1 mM of isopropyl β-D-thiogalactoside (IPTG, Sigma Aldrich) was added to induce protein production. After 16 h, cells were harvested, lysed by sonication and dissolved using urea. Thereafter, CD38A was obtained through the use of Nickel columns (Sigma Aldrich). The CD38A-containing fractions were pooled, refolded and used to immunize three independent mice. Animals were immunized intravenously (i.v.) at days 0, 15, 30, and 45. Mice were sacrificed 3 d after the last immunization, and splenocytes isolated. Generation of hybridomas by fusion of mouse splenocytes with SP2 myeloma cells, isolation of clones, and purification of specific anti-trout mAbs were performed as previously described (36 (link)). The recombinant CD38A protein was used to test the specificity of the antibodies by ELISA following methods previously described (37 (link)).