Specific DNA aptamer–immobilized cryogel membranes as novel bioaffinity supports and their potential for the purification of activated protein C

Abstract

Activated protein C (APC), a serine protease produced from zymogen protein C (PC), is the key enzyme of the protein C pathway. APC has anticoagulant, anti-inflammatory, and cytoprotective features. APC has recently been shown to significantly reduce coagulation as well as mortality in patients with severe sepsis. Herein, we aimed to develop an affinity support material that allows the purification of plasma APC for the first time. In this research, a novel APC-specific DNA aptamer–based poly(2-hydroxyethyl methacrylate-glycidyl methacrylate) (poly(HEMA-GMA/DNA-Apt)) macroporous cryogel membrane at different molar ratios was prepared using affinity binding method and their potential for purification and identification of APC was investigated. The DNA aptamer–immobilized cryogels were characterized to examine their structural and morphological properties. The effect of pH, initial concentration, temperature, ionic strength difference, and flow rate changes was examined. Selectivity studies were performed in the presence of APC and competitive proteins, and cryogel support materials were shown to have a very high affinity for APC. Adsorption capacity was found to be 89.02 mg/g. Finally, NaCl revealed efficiency for APC desorption and the reuse of cryogels was successfully tested for ten cycles.