Purification of Recombinant PON1 Proteins

After the plasmids were transformed into E. coli BL21 (DE3) cells, the PEP-1-PON1 and control PON1 proteins were induced by adding 0.5 mM isopropyl-β-D-thio-galactoside (Duchefa, Haarlem, Netherlands) at 37°C for 6 h. Recombinant proteins obtained from harvested cells were lysed by sonication after which the proteins were purified by Ni²⁺-nitrilotriacetic acid Sepharose affinity column chromatography (Qiagen, Valencia, CA, USA) and PD-10 column chromatography (Amersham, Brauncschweig, Germany) according to the manufacturer’s instructions [28] (link)–[36] (link). To remove endotoxins of proteins, purified control PON1 and PEP-1-PON1 were treated with a Detoxi-Gel™ endotoxin removing gel (Pierce, Rockford, IL, USA) as per manufacturer’s instructions [34] (link). The Bradford assay was used to estimate protein concentration [37] (link).