Purification and activation of AEPs

The lysate containing each expressed AEP was mixed with 3 mL of Talon^® metal affinity resin (Clontech) at 4 °C for 1 h, then loaded onto a gravity column. The column was washed with lysis buffer containing 20 mM imidazole, and the target protein was eluted with buffer containing 150 mM and 300 mM imidazole. The fractions were screened by SDS-PAGE and stained with InstantBlue (Expedeon).

To self-activate AEPs, fractions were desalted by a PD10 column (GE healthcare). Then, activation was performed at pH 4.0 with 0.1 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) at 37 °C for 30 min (MCoAEP2) or 2 h (all other AEPs).

MCoAEP2 was subsequently purified by cation exchange chromatography (GE Healthcare) with a linear gradient (0–100% buffer B, 50 mM sodium acetate, 1 M NaCl, 10% glycerol, pH 4.0), and the final product analyzed by SDS-PAGE followed by in-gel digestion for MS/MS confirmation. Purified MCoAEP2 was stored at -80 °C until further analysis.