The Mtb RelMtb 53 to 446 construct was purchased from GenScript USA Inc. (Piscataway, NJ, USA). Truncated RelMtb was PCR-amplified from genomic DNA and cloned into pET42b (Novagen, Darmstadt, Germany) using Nde l/Hind III restriction sites. The pET24b 6HisFLAG TB RelMtb 53 to 446 construct was amplified into E. coli’s top 10 cells (Thermo Fisher Scientific Inc.) using a heat shock procedure, and transformant colonies were screened by PCR. Positive clones were verified by DNA sequencing.

The recombinant protein was expressed in a strain of Rosetta 2 competent cells (Merck KGaA, Darmstadt, Germany) overnight. Following protein expression, the cells were harvested and sonicated in lysis buffer, and cell debris was removed by centrifugation at 13,000 rpm at 4°C for 20 min. Expression of soluble protein was monitored by Novex 4 to 20% Tris-Glycine Protein Gels (Thermo Fisher Scientific Inc.) in reducing running conditions and visualized by instant blue staining (Expedeon Inc., San Diego, CA, USA). Peptide mass fingerprinting (PMF) analysis gave a 49% coverage, which confirms the identity of the protein.

The supernatant was applied to a 1-ml HisTrap HP column (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) previously equilibrated in 50 mM Na2HPO4, 300 mM NaCl, and 20 mM imidazole. The protein was then eluted in three steps using ÄKTAxpress (GE Healthcare Bio-Sciences Corp.) with the equilibration buffer by increasing the concentration of imidazole (100, 250, and 500 mM). The chromatogram revealed two peaks that were dialyzed separately using a 10-kDa snake skin (Thermo Fisher Scientific Inc.) overnight at 4°C to remove imidazole. The P1 and P2 pools of His-FLAG-RelMtb were further submitted to size exclusion purification, and the supernatant was adjusted to 500 mM NaCl and 10% glycerol before it was applied to a 124-ml HiLoad Superdex 75 16/600 column (GE Healthcare Bio-Sciences Corp.) equilibrated in 50 mM tris base (pH 8.0), 500 mM NaCl, 1 mM dithiothreitol, and 10% glycerol. A similar pattern of three peaks was observed for the P1 and P2 pools. The large peaks obtained in each preparation were pooled together, yielding 108.1 mg of His-FLAG-RelMtb with good purity (>95%) by gel densitometry (Synoptics Ltd., Cambridge, UK) from 2 g of cell pellet. Protein concentration was determined by ultraviolet absorbance with a NanoDrop (Thermo Fisher Scientific Inc.) and aliquoted for storage at −80°C. The MW obtained by LC-MS analysis (46,539.5 Da) has an acceptable variation range with respect to the expected mass and indicates the loss of the N-terminal methionine (129.5 Da). These results have been confirmed by PMF analysis as well.

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