Expression and production of recIAP
In this study, cDNA for recombinant human intestinal alkaline phosphatase (recIAP) (Peters, 2016b; Kiffer-Moreira, 2014) was used to construct the expression vector pMH3-recIAP (Qian, 2010), which was then transferred into CHO-S cells (CVCL_7183, Life Technologies). The cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum (FBS) and subjected to G418 pressure screening to obtain stable, high-expression clones. These clones were expanded and cultured by mammalian cell bioreactors in order to obtain harvest medium for purification. The harvest medium was centrifuged and passed through a pyrogen-removed cation column (SP XK50,GE Healthcare) in order to remove heteroproteins. The flow-through containing recIAP fraction was collected and passed through the pyrogen-removing anion column (Q XK50, GE Healthcare) again to collect recIAP-containing elute. All the solutions used in the purification process were passed through a hollow fiber column (Borglong Biotechnology Co., Ltd.) to remove endotoxins The purity of concentrated recIAP solution was determined using SDS-PAGE electrophoresis and high-pressure liquid chromatography (HPLC). The recIAP solution was found to have 92.45% purity.