Ng gyrase isolation

Ng gyrase isolation was performed using a modification from Gross et al., 2003. S-tagged gyrA and His-tagged gyrB were cloned into NcoI/PstI and XhoI/BglII, sites respectively in pET-Duet1 and transformed into BLR(DE3). Growth and lysozyme treatments were as described above for topoisomerase IV (Pan and Fisher, 1999) and the supernatant was mixed with Ni-NTA resin and shaken overnight at 4 °C. The resin was loaded into a column and the flow through containing S-tagged gyrase A was further purified using an S-protein agarose (Sigma) by elution with 20 mM Tris pH 7.5, 300 mM NaCl, 3 M MgCl₂.

His-tagged gyrase B was loaded into a Ni-NTA column, washed with 20 mM Tris-HCl [pH 8.0], 300 mM NaCl, 10% glycerol, 0.1% Triton X-100, eluted in the same buffer containing 200 mM imidazole. The purified protein judged by SDS PAGE were pooled and diluted 32-fold in buffer D (50 mM Tris HCl [pH 8.0], 2 mM dithiothreitol (DTT), 1 mM EDTA, 10% glycerol).

Further purification of gyrase A and gyrase B used a mono-Q (GE Healthcare) column in buffer D and a linear gradient elution from 0 to 1 M NaCl. Fractions were analyzed by SDS PAGE and Coomassie blue staining.

The fractions containing the proteins were pooled, concentrated and desalted using Ultrafree-15 Biomax-100 membrane centrifugal filter units (Millipore) in 10 mM Tris pH 7.9, 50 mM KCl, 0.1 mM EDTA, 2 mM DTT. Glycerol was added to the purified protein at a final concentration of 25% and flash frozen in liquid nitrogen, and stored at –80 °C. The protein concentrations were determined by A280 nm and calculated extinction coefficients of Ng GyrA (56,400 M^–1 cm^-1) and Ng GyrB (51,520 M^–1 cm^–1). Active gyrase (A₂B₂) was formed prior to the start of an assay using equal molar amounts (15 μM GyrA and GyrB).