The expression and purification of glutathione-S-transferase tagged human IDE (GST-IDE in pGEX-6p-1 vector, kindly provided by Dr. Malcolm Leissring of the University of California at Irvine) were accomplished following a protocol26 (link) provided by Dr. Leissring with some modifications. Expression of GST-IDE in Escherichia coli BL21 (DE3) CodonPlus RIL was induced with 50 μM IPTG overnight at 25 °C. The non-metalloprotease inhibitor phenylmethylsulfonyl fluoride was added prior to cracking the cells. GST-IDE was purified using a 5 mL GST Trap Fast Flow column on an ÄKTA Pure FPLC (GE Healthcare Life Sciences) and eluted with PBS containing 10 mM glutathione. After cleavage of the GST tag using GST PreScission protease, further purification of IDE was accomplished using a HiLoad 16/600 Superdex S-200 pg gel filtration column preequilibrated with PBS on the ÄKTA Pure FPLC. Purified protein concentration was determined by UV–vis spectroscopy using the molar extinction coefficient of IDE at 280 nm, ε280nm = 113570 M−1 cm−1.42 (link) Flash-frozen IDE aliquots containing 1% glycerol (v/v) were stored at −80 °C in PBS (pH 7.4). Activity of IDE was confirmed using insulin (Sigma-Aldrich) or the fluorogenic peptide Mca-RPPGFSAFK(Dnp)-OH (R&D Systems) as substrates.