Hiload 16 60 200 pg

The sequence of the full-length Mot1 (1 – 1886) was isolated from the Chaetomium thermophilum cDNA library and cloned into pETDuet-1 vector (Novagen, Germany) harboring N-terminal His₆ tag followed by TEV cleavage site. CtMot1^ΔC(1 – 1836) was cloned into pET21 vector containing PreScission protease cleavage site and C-terminal His₆ tag. Both constructs were expressed in Escherichia coli Rosetta(DE3) cells (Novagen) and purified using Ni²⁺-NTA agarose (QIAGEN, Germany). After proteolytic cleavage of the expression tags, the proteins were further purified using ion-exchange chromatography (HiTrap Q HP, GE Healthcare, Germany) and size exclusion chromatography (HiLoad 16/60 200 pg, GE Healthcare). Proteins were concentrated to ~15 mg/ml in 20 mM Tris pH 7.5, 50 mM NaCl and 15% glycerol and stored at −80°C. Selenomethionine labelling of CtMot1^∆C was performed in E. coli B843 (Novagen) using SelenoMethionine Medium Base and Nutrient Mix (Molecular Dimensions, UK) supplemented with L(+)-Selenomethionine (Acros Organics, Germany) at 42 mg/L. Purification of selenium-derivatized protein was performed according to the same protocol as for the native protein.

Residues 1–326 of MPK38 were subcloned into the pET-21b vector (Novagen). The MPK38 (T167E) mutant, which mimics a constitutively phosphorylated state, was generated using the QuikChange kit (Stratagene). The MPK38 (T167E) construct was transformed into Escherichia coli BL21(DE3) cells. The expression of MPK38 (T167E) was induced by 0.5 mM IPTG at 18°C for 16 h. The cells were harvested and suspended in cell-lysis buffer (20 mM Tris–HCl pH 7.5, 500 mM NaCl). The cells were lysed by sonication and the supernatant was separated by centrifugation. The cell supernatant was applied onto a HisTrap HP column (GE Healthcare) and washed with washing buffer (20 mM Tris–HCl pH 7.5, 500 mM NaCl, 50 mM imidazole). MPK38 (T167E) was eluted with elution buffer (20 mM Tris–HCl pH 7.5, 500 mM NaCl, 200 mM imidazole). MPK38 (T167E) was purified using a HiTrap Q anion-exchange column (GE Healthcare). Finally, MPK38 (T167E) was eluted by gel-filtration chromatography (HiLoad 16/60 200 pg, GE Healthcare) in size-exclusion chromatography (SEC) buffer (20 mM Tris–HCl pH 7.5, 300 mM NaCl, 5 mM DTT).