Isopropyl β d thiogalactopyranoside iptg

Unless stated otherwise all chemicals used were purchased from Sigma Aldrich (Saint Louis, MO, USA); all DNA ladders, restriction enzymes and their buffers were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
Primers for DNA amplification and sequencing were synthesized by Biolegio (Nijmegen, the Netherlands) or Integrated DNA technologies (Coralville, IA, USA). Sequencing services for all DNA constructs were provided by Macrogen (Amsterdam, the Netherlands). pGEM-T Easy cloning system I was purchased from Promega (Fitchburg, WI, USA). pET28a vector was purchased from Novagen (Darmstadt, Germany). EDTA-free protease inhibitor cocktail tablet was obtained from Roche diagnostics (Basel, Switzerland). n-dodecyl-β-D-maltopyranoside (DDM) was obtained from Anatrace (Maumee, OH, USA). Precision Plus Protein standards were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Isopropyl-β-D-thiogalacto- pyranoside (IPTG) was purchased from Thermo Fisher Scientific. ΔslyD BL21 (DE3) E. coli strain was a kind gift from Prof. Ry Young (Dept. Biochemistry and Biophysics, Texas A&M University, USA).

The recombinant (PspA_1-5c+p -pET28a) vector was transformed into the chemically prepared competent E. coli BL21 (DE3) cell via heat shock transformation. Positive clones were recognized by restriction enzyme digestion and colony PCR with universal T7 primers (https://www.addgene.org/mol-bio-reference/sequencing-primers/). Expression of the recombinant PspA_1-5c+p was induced by adding 1 mM Isopropyl-β-D, Thiogalactopyranoside (IPTG) (Thermo Fisher Scientific, USA) in Luria–Bertani Broth (LB) medium (Sigma Aldrich, USA) at 37 °C and incubated for 16 h, then was evaluated by 12% SDS-PAGE, and using HRP-conjugated anti-His tag antibody (Sigma, USA) confirmed by western blot analysis. By the manufacturer's instructions (Qiagen, Hilden, Germany), the recombinant PspA_1-5c+p was purified using a Ni–NTA column (Qiagen, Hilden, Germany) under native conditions. The purified PspA_1-5c+p was dialyzed with dialysis tubing (cutoff 12KDa) overnight at 4 °C against PBS (Sigma, USA) and measured by the Bradford protein assay [47 (link)]. A Limulus amebocyte lysate (LAL) test was done to assess LPS contamination using the LAL kit (Lonza QCL-1000 ®, Basel, Switzerland).

E. coli strain BL21(DE3) was transformed with pET-28a-SH3-I₂-GUK::GFP, pET-28a-SH3-I₃-GUK::GFP and with pET-28a-CaM::mCherry plasmid DNA. The cells were grown at 37 °C to an optical density of 0.8 at 600 nm and protein expression was induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside ((IPTG) Thermo Fisher Scientific, USA) overnight. The next day, the cells were centrifuged at 4000 x g for 10 min and the pellets were stored at −80 °C. The bacterial cells were lysed by sonication (Vibra cell, Sonics) with Tris buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, imidazole and protease inhibitor (Roche, Switzerland)). As the proteins were predominantly present in the soluble fraction, the sonicated samples were centrifuged at 30,000 x g for 1 h to remove cellular debris. The proteins were purified by Ni^2+-nitrilotriacetic acid (NTA)-agarose affinity chromatography. The supernatant was passed through Ni^2+-NTA-agarose resin (Qiagen) and the protein-bound beads were washed with 4 column volumes (~25 ml) of wash buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 50 mM imidazole. 4 ml elution buffer (10 mM Tris-Cl (pH 7.4), 150 mM NaCl and 500 mM imidazole) was added to elute the target proteins. The eluted proteins were concentrated to 500 μl (Vivaspin-Turbo4, 30 kDa) and was ultracentrifuged at 35,000 x g at 4 °C for 30 min to pellet out the protein aggregates.