Recombinant Expression and Purification of AspH

N-Terminally His₆-tagged human AspH_315–758 (His₆-AspH_315–758) was produced in Escherichia coli BL21 (DE3) cells using a pET-28a(+) vector and purified by Ni(II)-affinity chromatography (HisTrap HP column, GE Healthcare; 1 mL/min flow rate) and size-exclusion chromatography (HiLoad 26/60 Superdex 75 pg 300 mL column; 1 mL/min) using an ÄKTA Pure machine (GE Healthcare), as previously reported.28 (link), 30 (link) AspH was > 95% pure by SDS-PAGE and MS analysis and had the anticipated mass (m/z calculated for His₆-AspH_315–758: 54519 Da, found: 54519 Da). Purified AspH was stored in 50 mM HEPES buffer (pH 7.5, 150 mM NaCl) at a concentration of 125 μM at −78 °C; fresh aliquots were used for all AspH inhibition assays.