Hiload superdex 75pg

CaM-WT and E140G variant were expressed and purified as previously described (79 (link)). In short, CaM pESUMOPro-Kan plasmids were transformed into Escherichia coli BL21(DE3) STAR and cultured in 2xYT media containing 100 μg/ml kanamycin. Expression was induced with 0.5 mM IPTG overnight at 18 °C. Cells were harvested by centrifugation and pellets were resuspended in 50 mM Hepes, 200 mM NaCl, pH 7.5 supplemented with protease inhibitor cocktail (Proteoloc, Abcam). Cells were lysed with lysozyme (1 mg/ml) for 30 min on ice followed by sonication. Lysates were further treated with BaseMuncher (Abcam) and clarified by ultracentrifugation.
Clarified lysates were purified on a HisTrap HP column (ÄKTA Start, GE Healthcare) using a linear gradient of 0 to 500 mM imidazole. Eluted proteins were dialyzed overnight at 4 °C (8 kDa) to remove the imidazole, and His-tag was removed by treatment with SUMO protease (ULP1). CaM proteins were then further purified by reverse HisTrap and size-exclusion chromatography (HiLoad Superdex 75pg, ÄKTA Pure, GE Healthcare). Fractions containing the purified proteins were concentrated using Amicon centrifugation units (3 kDa), flash-frozen in liquid nitrogen, and stored in -80 °C until used.

The variants of PTPN4 named F620G, Q621G, Y52G, I623G and ΔFQYI were obtained by using the Quik-Change Mutagenesis kit (Stratagene) according to the manufacturer’s instructions, and the substitutions were confirmed by DNA sequencing. Template DNA used for PTPN4 mutants was the PDZ-PTP_WT construct cloned in a pET15b expression plasmid. The PTP construct was obtained by using the Pwo polymerase (Roche) according to the manufacturer’s instructions, and the insertion was confirmed by DNA sequencing. Template DNA used for the PTP construct was a synthetic construct comprising the PTP domain cloned in a pgex6p1 expression plasmid. Expression and purification of linker-PTP and PTP construct were performed as described previously^{13 (link)}. Bidomain PDZ-PTP variants were expressed and purified as previously described without TEV cleavage. Briefly, clarified supernatants were loaded on Ni²⁺ column (HiTrap Chelating HP, GE), washed and eluted with an imidazole gradient from 40 mM to 1 M. The eluted fractions containing the protein were pooled and loaded on a gel filtration column (Hiload Superdex 75 pg, GE). Proteins were concentrated using centrifugal filter devices (Vivaspin, Sartorius). Protein concentration was estimated from its absorbance at 280 nm. The peptide Cyto8-RETEV (SAESHKSGRETEV) was synthesized in solid phase using a Fmoc strategy (JPT).