Cloning, high level expression of human paraoxonase-3 in Sf9 cells and pharmacological characterization of its product.

Human paraoxnase-3 (hPON3) (EC3.1.8.1) is a lipid-associated enzyme with antioxidant activity, and can inhibit the oxidation of low-density lipoprotein (LDL), thereby inhibiting early atherogenic process. In the present study, human PON3 gene was cloned from Human Fetal Liver Marathon-Ready cDNA and expressed in insect cells using baculovirus vector. Twenty-eight milligrams of purified recombinant hPON3 (rhPON3) was obtained from 1L Sf9 cells culture. The Km and Vmax values of rhPON3, with respective to phenylacetate hydrolysis were 7.46+/-4.40 mM and 89+/-10.54 U/mg (n=3). The kinetic parameters of Vmax and Km for dihydrocoumarin hydrolysis by rhPON3 were 698+/-248 U/mg and 0.84+/-0.24 mM (n=3). LDL oxidation assay indicated that rhPON3 could effectively protect LDL against copper-induced oxidation in vitro.