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.