High-level expression of recombinant human paraoxonase 1 Q in silkworm larvae (Bombyx mori).
Human serum paraoxonase 1 (hPON1) belongs to a family of enzymes that catalyze the hydrolysis of a broad range of esters and lactones. Although the very first identification of hPON1 might have been as a calcium-dependent paraoxonase/arylesterase, PON1 is in fact a lactonase associated with high-density lipoprotein and strongly stimulated by apoA-I. PON1 hydrolyzes various organophosphates, including insecticides and nerve gases. PON1 also plays a key role in prevention of atherosclerosis. Mediation of cholesterol efflux from macrophage is a key in vivo function of PON1. In present study, the hPON1 Q gene was cloned into baculovirus transfer vector pVL1392 and expressed in silkworm expression system. The rhPON1 Q presented two bands with every near molecular weight of about 40 and 43 kDa according to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis. The expression level was up to 1,256 mg/L in haemolymph, about 50 times as high as that from BmN cells (24.8 mg/L). After purified by two chromatography steps (DEAE-Sepharose and HiTrap Chelating HP), the purity of rhPON1 Q was up to 90%, and the enzymatic properties are similar to serum hPON1.
Zhu
Jie
J
School of Life Science and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, 210093, People's Republic of China.
Ze
Yuguan
Y
School of Life Science and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, 210093, People's Republic of China.
Zhang
Chi
C
School of Life Science and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, 210093, People's Republic of China.
Zang
Yuhui
Y
School of Life Science and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, 210093, People's Republic of China.
Lu
Haiqin
H
School of Life Science and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, 210093, People's Republic of China.
Chu
Ping
P
School of Life Science and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, 210093, People's Republic of China.
Sun
Manji
M
Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing, 100850, People's Republic of China.
Qin
Junchuan
J
School of Life Science and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, 210093, People's Republic of China. jcqin@nju.edu.cn.
D000818:Animals; D030801:Animals, Genetically Modified; D043303:Aryldialkylphosphatase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D012831:Bombyx / Q000235:genetics / Q000378:metabolism*; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006458:Hemolymph / Q000737:chemistry; D006801:Humans; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human