Pgex 4t

For Figure 5A, the nda3-KM311 strain was cultured at 30°C for 18 h and then shifted to 17°C for 13 h in YE5S to induce mitotic arrest. Cells were collected and lysed with glass beads in HB buffer. Cell extracts were incubated with agarose-conjugated p13/suc1 (Millipore, Billerica, MA) for 1.5 h at 4°C. After washing with HB buffer, the pellet was resuspended with HB buffer and used as the CDK solution.
For the assay in Figure 5C, The Cdc2-GST fusion protein was purified from S. pombe cells using a GST pull-down method. First, the cdc2⁺ gene was tagged with the GST gene using the standard method (Bähler et al., 1998 (link)) in the nda3-KM311 strain. The resultant strain, nda3-KM311 cdc2-GST, was cultured at 30°C for 18 h and then shifted to 17°C for 13 h. Cell extraction was done as described. Crude extracts were then incubated with glutathione Sepharose 4B (GE Healthcare) for 1.5 h at 4°C. After washing with HB buffer, the pellet was resuspended with HB buffer and used as the CDK solution. Activity of the purified Cdc2-GST was confirmed by an in vitro kinase assay using histone H1 (Calbiochem, San Diego, CA) as a substrate.
To express recombinant GST–Alp7-WT and GST–Alp7-5A in E. coli BL21 (DE3) cells, coding sequences for alp7⁺ and alp7-5A were amplified with PCR and cloned into the GST-containing vector pGEX-4T (GE Healthcare).

Wild-type and mutant adf genes from T. gondii were cloned into pGEX4T (GE Healthcare, Uppsala, Sweden) and expressed as glutathione S-transferase fusion proteins in Escherichia coli strain BL21 (DE3; Life Technologies). Recombinant proteins were purified as previously described (Wong et al., 2011 (link)) and dialyzed against PBS before cleavage with thrombin protease (GE Healthcare). Untagged ADF proteins were purified by size exclusion chromatography using a Superdex 200, GL10/300 column (GE Healthcare) and eluted in 20 mM MES/10 mM NaCl, pH 7.0, for downstream biochemical analysis.