Recombinant mouse ameloblastin (rAmbn) and the variant delta 5 (AmbnΔ5), which lacks the sequence encoded by Exon 5, were expressed and purified following the method described in our recent publication (Su et al. 2019b (link)). Briefly, Ambn was expressed in Escherichia coli with Histidine (His), Thioredoxin (Trx), and S-tags in BL21, using pET-32a plasmid (Novagen) with inserted mouse Ambn gene (GenBank No. AAB93765.1). The expressed protein was purified using Nickel affinity chromatography (Ni-NTA Agarose, Qiagen), followed by dialysis through 10K MWCO dialysis membrane (SnakeSkin™ Dialysis Tubing, ThermoFisher). His-, Trx-, and S-tags were cleaved by Enterokinase (light chain, New England Biolabs), and were removed by a reversed phase High Performance Liquid Chromatography (HPLC) system (Varian Prostar system with Agilent OpenLab CDS software). Phenomenex C4 column (10 × 250 mm, 5 μm) was used, and proteins were eluted with an increasing gradient of 32–72% acetonitrile over 80 min, at a flow rate of 1.5 ml/min. Collected proteins were lyophilized and characterized with SDS-PAGE. The heat denaturation of rAmbn was performed by heating rAmbn at 95°C for 10 min.