title: Expression and purification of biologically active recombinant human paraoxonase 1 from a Drosophila S2 stable cell line.
abstract: Many pesticides and chemical warfare nerve agents are highly toxic organophosphorus compounds (OPs), which inhibit acetylcholinesterase activity. Human paraoxonase 1 (PON1) has demonstrated significant potential for use as a catalytic bioscavenger capable of hydrolyzing a broad range of OPs. However, there are several limitations to the use of human PON1 as a catalytic bioscavenger, including the relatively difficult purification of PON1 from human plasma and its dependence on the presence of hydrophobic binding partners to maintain stability. Therefore, research efforts to efficiently produce recombinant human PON1 are necessary. In this study, we developed a Drosophila S2 stable cell line expressing recombinant human PON1. The recombinant human PON1 was fused with the human immunoglobulin Fc domain (PON1-hFc) to improve protein stability and purification efficiency. We purified the recombinant human PON1-hFc from the S2 stable cell line and characterized its enzymatic properties for OP hydrolysis. We purified the recombinant human PON1-hFc from the S2 stable cell line and characterized its enzymatic properties for OP hydrolysis compared with those of the recombinant human PON1 derived from E. coli. We observed that the recombinant human PON1-hFc is functionally more stable for OP hydrolyzing activities compared to the recombinant human PON1. The catalytic efficiency of the recombinant PON1-hFc towards diisopropyl fluorophosphate (DFP, 0.26 × 106M-1min-1) and paraoxon hydrolysis (0.015 × 106M-1min-1) was 1.63- and 1.24-fold higher, respectively, than the recombinant human PON1. Thus, we report that the recombinant PON1-hFc exerts hydrolytic activity against paraoxon and DFP.
authors.affiliation: Department of Microbiology and Molecular Biology, Chungnam National University, Daejeon 34134, South Korea.
authors.forename: Hyeongseok
authors.initials: H
authors.lastname: Yun
authors.affiliation: Department of Microbiology and Molecular Biology, Chungnam National University, Daejeon 34134, South Korea.
authors.forename: Jiyeon
authors.initials: J
authors.lastname: Yu
authors.affiliation: Department of Microbiology and Molecular Biology, Chungnam National University, Daejeon 34134, South Korea.
authors.forename: Sumi
authors.initials: S
authors.lastname: Kim
authors.affiliation: Department of Microbiology and Molecular Biology, Chungnam National University, Daejeon 34134, South Korea.
authors.forename: Nari
authors.initials: N
authors.lastname: Lee
authors.affiliation: Department of Microbiology and Molecular Biology, Chungnam National University, Daejeon 34134, South Korea.
authors.forename: Jinhee
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Microbiology and Molecular Biology, Chungnam National University, Daejeon 34134, South Korea.
authors.forename: Sungrae
authors.initials: S
authors.lastname: Lee
authors.affiliation: New Drug Development Center, DGMIF, 80, Cheombok-ro, Dong-gu, Daegu 41061, South Korea.
authors.forename: Nam Doo
authors.initials: ND
authors.lastname: Kim
authors.affiliation: Agency for Defense Development, P.O. Box 35, Yuseong, Daejeon 34186, South Korea.
authors.forename: Chiho
authors.initials: C
authors.lastname: Yu
authors.affiliation: Department of Microbiology and Molecular Biology, Chungnam National University, Daejeon 34134, South Korea. Electronic address: jrrho@cnu.ac.kr.
authors.forename: Jaerang
authors.initials: J
authors.lastname: Rho
mesh_terms: D000818:Animals; D043303:Aryldialkylphosphatase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002460:Cell Line; D004331:Drosophila melanogaster; D015870:Gene Expression; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human
keywords: Bioscavenger; Diisopropylfluorophosphate; Organophosphorus compound; Paraoxon; Paraoxonase 1