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.
Lu
Haiqin
H
School of Life Science and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People's Republic of China.
Zhu
Jie
J
Zang
Yuhui
Y
Ze
Yuguan
Y
Qin
Junchuan
J
D000818:Animals; D043303:Aryldialkylphosphatase; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000235:genetics* / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D018411:Spodoptera
D017931:DNA Primers; D011994:Recombinant Proteins; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; C019734:arylesterase; D043303:Aryldialkylphosphatase; C113656:PON3 protein, human