Expression and purification of rTNT protein

The TNT gene was excised from TNT-pUC57 construct and cloned into dephosphorylated SnaBI-cut pMTSA expression vector. Upon induction, TNT-pMTSA construct produces C-terminal hexa-Histidine tagged-TNT protein. To purify the TNT protein, TNT-pMTSA plasmid containing E. coli C43 (DE3) cells were grown in glucose supplemented LB medium. At mid-log stage, cells were induced with 0.2% L-arabinose and incubated at 25 °C with constant shaking for 10 h. The induced cells were pelleted and washed with PBS buffer (4.3 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 150 mM NaCl, 2.7 mM KCl, pH 7.4) and resuspended in buffer for lysing cells (2 mM PMSF in PBS, pH 8.0). After sonication the cell lysate was centrifuged and the pellet fraction solubilised in buffer (PBS buffer with 8 M urea, pH 8.0) by continuous shaking at 25 °C for 12 h. Filtered soluble fraction obtained after centrifugation of the solubilised sample was incubated with Ni-NTA resin for binding (Qiagen, Germany) at 25 °C overnight. Ni-NTA bound protein was eluted using imidazole gradient (8 M urea in PBS buffer with 20 mM to 500 mM imidazole, pH 8.0). The pooled fractions were dialysed against buffer (PBS, pH 8.0, 8-0 M urea) to gradually remove urea. To assess purity, dialysed rTNT protein was run on an SDS-PAGE (Supplementary Fig. 2c). Antibodies were raised against the purified TNT protein (Supplementary methods).