Cloning, purification, and refolding of human paraoxonase-3 expressed in Escherichia coli and its characterization.
Human paraoxonase (hPON3) is a high density lipoprotein-related glycoprotein with multi-enzymatic properties and antioxidant activity which is proposed to participate in the prevention of low density lipoprotein (LDL) oxidation. In this study, hPON3 gene was amplified from Human Fetal Liver Marathon-Ready cDNA and expressed in Escherichia coli. A majority of the expressed protein existed as inclusion bodies. The inclusion bodies were solubilized with Triton X-100 and refolded in vitro. The refolded rhPON3 was purified by DEAE-Sepharose Fast Flow and its purity was up to 90%. The Km and Vmax values of refolded rhPON3, in respect to phenylacetate hydrolysis were 7.47 +/- 2.14 mM and 66 +/- 17 U/min/mg (n = 3). The Km and Vmax values of refolded rhPON3, in respect to dihydrocoumarin hydrolysis were 0.83 +/- 0.21 mM and 621 +/- 66 U/min/mg (n = 3). The refolded rhPON3 exhibited similar antioxidant activity to that of rhPON3 purified from the soluble fraction of cell lysate and could effectively protect LDL from Cu2+ induced oxidation.
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
D043303:Aryldialkylphosphatase; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000378:metabolism; D008099:Liver / Q000196:embryology / Q000201:enzymology; D016366:Open Reading Frames; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D008077:Lipoproteins, LDL; D011994:Recombinant Proteins; C099252:oxidized low density lipoprotein; D004950:Esterases; D043303:Aryldialkylphosphatase; C113656:PON3 protein, human