Recombinant AMA1 Cytoplasmic Tail Purification

Recombinant AMA1 cytoplasmic tail protein (residues 567–622) with a thrombin-cleavable GST fusion (GST-AMA1_cyt), or an AMA1 Ser₆₁₀Ala point mutant of the same protein (GST-AMA1_cyt _S610A), was expressed in Escherichia coli BL21 and purified as described previously [23 (link)]. For NMR experiments, labelled protein was produced using E. coli grown in M9 medium containing ¹⁵N ammonium sulphate and ¹³C glucose as the sole nitrogen and carbon sources. To cleave the GST component, 100 μg GST-AMA1_cyt protein was treated with one unit of human alpha-thrombin (HTI) overnight at 18°C in 50 mM Tris-HCl, pH 8.2, and 2 mM CaCl₂. Following the digestion, glutathione agarose (Sigma) was used in excess to trap GST in solution and was later removed by centrifugation. The protein solution was then passed through a Superdex 75 HR 26/60 column equilibrated with 20 mM Tris-HCl, pH 8.2, and 150 mM NaCl to remove thrombin and residual GST. For phosphorylation, 200 μg of AMA1_cyt in 20 mM Tris-HCl, pH 8.2, and 150 mM NaCl was treated with 3.5 μg of mouse PKAc-α (Bioaffin GmbH & Co KG) in the presence of 2 mM ATP, 20 mM MgCl2, and 2 mM DTT and incubated overnight at 30°C. The protein solution was then passed through a Superdex 75 HR 26/60 column equilibrated in 20 mM Tris-HCl, pH 8.2, and 150 mM NaCl. This step completely removed PKAc-α.