Ni nitrilotriacetic acid nta column

M. tuberculosis σ^A, σ^B, and RbpA were cloned in pET21a with a C-terminal His tag, transformed into E. coli BL21(DE3)pLysS, grown to an A₆₀₀ of 0.4, and induced with 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) at 30°C. The cells were lysed in a buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 5% glycerol, and the clarified lysate was loaded on an Ni-nitrilotriacetic acid (NTA) column (Qiagen). Nonspecifically bound proteins were removed by washing with lysis buffer containing 40 mM, 35 mM, and 20 mM imidazole, and the protein was eluted with 150 mM imidazole. For purification of M. tuberculosis RNA polymerase, BL21(DE3)pLysS was cotransformed with pETDuet-Mtbββ′ and pRsfDuet-Mtbαω, grown at 30°C to an A₆₀₀ of 0.4, and induced with 0.4 mM IPTG at 16°C for a period of 18 h. The cells were lysed by sonication and passed through an Ni-NTA column (Qiagen) that had been equilibrated with 50 mM Tris, 300 mM NaCl, and 5% glycerol (lysis buffer). The column was washed with lysis buffer and 40 mM imidazole and eluted with lysis buffer and 150 mM imidazole. Fractions containing RNAP were loaded on a heparin-Sepharose matrix (GE Healthcare) that had been equilibrated with 50 mM Tris, 300 mM NaCl, and 5% glycerol and eluted with a buffer containing 1 M NaCl.

E. coli Rosetta(DE3)pLysS was transformed with pET21d-pprA and grown in 2× YT medium (Bio101, Inc.) supplemented with ampicillin (100 µg/ml). When the cell culture reached an A₆₀₀ of 1, PprA production was induced with 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside; Sigma) for 4 h at 37°C. Cells were harvested by centrifugation, suspended in 40 ml of buffer A (200 mM NaCl, 20 mM Tris-HCl, pH 7.5), and stored overnight at −20°C. Cell lysis was completed by sonication (probe-tip sonicator; Branson). The His-tagged PprA protein was purified on a Ni-nitrilotriacetic acid (NTA) column (Qiagen, Inc.), eluted with 200 mM imidazole in buffer A, and loaded onto a Superdex 200 column (Amersham Pharmacia Biotech) equilibrated against the same buffer. The PprA protein was concentrated using Vivaspin centrifugal concentrators with a nominal molecular weight limit cutoff of 5,000 (Vivascience), flash frozen in liquid nitrogen, and stored at −80°C.

Overnight (O/N) cultures of the expression strain E. coli BL21(DE3) or Rosetta(DE3)/pLysS containing the constructs were subcultured in fresh LB (dilution 1:20) and subsequently incubated at 37°C at 180 rpm until the optical density at 600 nm (OD₆₀₀) reached 0.5 to 0.8, at which point 0.5 or 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) (Sigma, Saint-Louis, MO, USA) was added. The cultures were then incubated at 28°C for 6 h or at 22°C O/N. The bacteria were then harvested by centrifugation (4,000 × g, 15 min, 4°C) and stored at −20°C. For purification, the bacterial pellet was resuspended in lysis buffer (50 mM NaH₂PO₄ · H₂O, 300 mM NaCl, 10 mM imidazole, 1 mg/mL lysozyme [pH 8]) supplemented with a protease inhibitor cocktail (1× Sigmafast inhibitor cocktail tablet, EDTA free; Sigma) and incubated at 10°C for 45 min. Three units per milliliter of Benzonase nuclease (Sigma) was added to reduce viscosity, and the soluble proteins were recovered by centrifugation at 10,000 × g for 30 min at 4°C. The clear lysate was filtered on 0.45-μm-pore filter (VWR, Oud-Heverlee, Belgium), and the protein was purified using Ni-nitrilotriacetic acid (NTA) columns (Qiagen, Hilden, Germany) following the manufacturer’s recommendations. The purity was assessed by SDS-PAGE (Bio-Rad, Hercules, CA, USA), and the protein concentration was established by a Bradford assay (63 (link)).