title: Expression, purification, and characterization of anti-plumbagin single-chain variable fragment antibody in Sf9 insect cell.
abstract: Plumbagin (PL; 5-hydroxy-2-methyl-1, 4-naphthoquinone) is an important secondary metabolite, mainly produced in the Plumbago zeylanica L. (Plumbaginaceae). A single-chain variable fragment (scFv) antibody, fusion of the variable regions of the heavy chain and light chain of immunoglobulin against PL (PL-scFv) was expressed by Bac-to-Bac Baculovirus Expression System using Spodoptera frugiperda (Sf9) insect cells and characterized to investigate potential use of PL-scFv as a tool for plant immunomodulation. Functional PL-scFv expressed in the Sf9 insect cells were purified using cation exchange chromatography followed by immobilized metal ion affinity chromatography (IMAC). The yields of the purified PL-scFv in the culture supernatant and Sf9 insect cells were 2.0 mg and 5.2 mg per 1 liter of Sf9 culture medium, respectively. Recombinant purified PL-scFv was then characterized by the indirect competitive enzyme-linked immunosorbent assay (ELISA). The cross-reactivity and sensitivity of PL-scFv expressed in Sf9 insect cells were compared with PL-scFv expressed in Escherichia coli and its parental anti-plumbagin monoclonal antibody (MAb 3A3) secreted from hybridoma cells. Intriguingly, the specificity of the PL-scFv expressed in Sf9 insect cells was found to be different from that expressed in E. coli and parental MAb 3A3, although the detectable level (0.2-25 μg/mL) was the same in ELISA using each antibody. Even more interestingly, the characteristics of PL-scFv, which have wide cross-reactivity against 1,4-napththoquinone, suggest its potential use as a tool for plant immunomodulation not only for breeding Plumbaginacea family containing PL but also for breeding other medicinal plants containing bioactive naphthoquinones.
authors.affiliation: Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Seiichi
authors.initials: S
authors.lastname: Sakamoto
authors.forename: Futoshi
authors.initials: F
authors.lastname: Taura
authors.forename: Ryota
authors.initials: R
authors.lastname: Tsuchihashi
authors.forename: Waraporn
authors.initials: W
authors.lastname: Putalun
authors.forename: Junei
authors.initials: J
authors.lastname: Kinjo
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
authors.forename: Satoshi
authors.initials: S
authors.lastname: Morimoto
mesh_terms: D000818:Animals; D016367:Baculoviridae; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003429:Cross Reactions / Q000276:immunology; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005822:Genetic Vectors / Q000276:immunology; D015394:Molecular Structure; D009285:Naphthoquinones / Q000737:chemistry / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000302:isolation & purification*; D018411:Spodoptera
chemical_list: D017931:DNA Primers; D009285:Naphthoquinones; D057127:Single-Chain Antibodies; C014758:plumbagin