**2.2. Recombinant expression and purification of** *Tb***CK**

Large-scale recombinant expression and purification of *Tb*CK was conducted using the con‐ struct pET-15bTEV-*Tb*CK in BL21 Rosetta (DE3) cells as described previously [23], except the cells were grown in tryptone phosphate broth [37], harvested by centrifugation at 3500 g for 20 min at 4°C and affinity purified with either a HisTrap™ FF crude column (enzyme activity assay) or a HisTALON Cartridge (thermal shift analysis).

Briefly, pelleted cells were suspended in buffer A (50 mMTris/HCl, pH 8.0, 300 mMNaCl and 10 mM imidazole) and lysed in the presence of DNase I by sonication. The lysate was cleared by centrifugation at 35000 g for 30 min at 4°C and applied to a 1 ml HisTrap™ FF crude column column (GE Healthcare) pre-loaded with Ni2+. Unbound proteins were re‐ moved by washing the column with 15 column volumes of buffer A containing 32.5 mM imidazole and *Tb*CK was eluted with 250 mM imidazole in the same buffer. Using a PD10 column, *Tb*CK was buffer exchanged into 50 mMTris/HCl, pH 8.0, 300 mMNaCl, glycerol (15% w/v) and stored at -80°C.

Alternatively, pelleted cells were suspended in 50 mMTris/HCl, pH 8.0, 300 mMNaCl and 5 mM imidazole and lysed by sonication. The lysate was cleared by centrifugation at 35000 g for 30 min at 4°C and applied to a 1 ml HisTALON Cartridge (Clontech).Unbound proteins were removed by washing the column with 10 column volumes of loading buffer, *Tb*CK was eluted with 15 mM imidazole and a final clearing wash of 250 mM imidazole in the same buffer. Using a PD10 column, *Tb*CK was buffer exchanged into 50 mM HEPES pH 8.0, 300 mMNaCl and 15% glycerol prior to storage at -80°C.

Typical yields were > 10 mg per litre of bacterial culture,*Tb*CK was stable and freeze thaw‐ ing did not lead to any significant loss of activity.
