Recombinant Expression and Purification of TtsA

The coding sequence for ttsA was amplified from S. Typhi strain ISP2825 and cloned by Gibson cloning strategy ^{49 (link)} into the expression vector pET28a+ (Novagen) resulting in N-terminal his-epitope tagged TtsA. Expression and purification of TtsA^WT and catalytic mutant TtsA^E14A were carried out as previously described ^{11 (link)}. Escherichia coli strain BL21 carrying the different plasmids were grown in LB containing kanamycin (50 μg/ml) to an OD_{600 nm} of 0.6–0.7 at 37°C. Expression of TtsA was subsequently induced by the addition of 0.5 mM IPTG, and induced cultures were incubated overnight at 25°C. Bacterial cells were pelleted by centrifugation, resuspended in lysis buffer [Tris-HCl (150 mM, pH 8.0), NaCl (100 mM), imidazol (10 mM), lysozyme (100 μg/ml), DNAse (100 μg/ml), saturated PMSF] and lysed with a French press. Lysates were subsequently pelleted (20,000g 1h, 4°C), and affinity-purified using a Nickel-resin (Qiagen) column. The eluates were diluted in 20 mM Tris-HCl, pH 8.0 buffer and loaded onto a Hi Trap Q ion-exchange column. Fractions from the ion-exchange chromatography were monitored on SDS–PAGE, concentrated, and further purified by using a Superdex 200 column. Final fractions were examined for purity on a 12 % SDS–PAGE.