Purification of Recombinant CDPK1 Enzymes

Full-length T. gondii wild type or Gly 128 Met (G128M) mutant CDPK1 enzymes were expressed with a C-terminal His tag in pET22b(+), as described previously^{12 (link)}. For protein purification, CDPK1 was expressed BL21 (DE3)V2RpAcYc-LIC+LamP E coli, which contains the LamP phosphatase. As a control, we also expressed cDNA fragment encoding human Src (hSrc) kinase domain in BL21, as described previously^{20 (link)}. Following overnight growth in Terrific Broth at 37°C, bacterial cultures were diluted 1:100 and grown for 3 hr at 37°C (O.D. 0.6–0.8), then induced with 0.3 mM IPTG during overnight growth at 15°C or 30°C. Cells were lysed using CellLytic B cell Lysis reagent (Sigma-Aldrich) and soluble proteins purified using HIS-select Nickel Affinity Gel. Purified proteins were dialyzed against storage buffer (i.e. 50 mM Tris-HCL, pH7.5, 150 mM NaCl), and stored in 25% glycerol containing 0.5 mM DTT at −80°C. Protein purity and concentrations were determined by SDS-PAGE and staining with Coomassie Blue (Invitrogen).