title: Construction and expression of specificity-improved single-chain variable fragments against the bioactive naphthoquinone, plumbagin.
abstract: We constructed a single-chain variable fragment (scFv) antibody against plumbagin (PL) with improved specific binding to PL. Variable heavy- and light-chain genes were cloned directly from the cDNA of hybridoma cell line 3A3 and assembled using the splice-overlap extension polymerase chain reaction (SOE-PCR) with specific primers including flexible peptide (Gly(4)Ser)(3) linker primers. The constructed scFv gene was ligated into the pET28a expression vector and transformed into Escherichia coli BL21 (DE3). The denatured protein expressed as inclusion bodies in E. coli was solubilized, purified, and refolded by a stepwise dialysis. Intriguingly, the refolded scFv against PL displayed higher PL-binding specificity than that of its parental monoclonal antibody, MAb 3A3, which suggests the possibility of improving the function by constructing the scFv antibody. These notable properties of the recombinant antibody against PL made it possible to develop an enzyme-linked immunosorbent assay (ELISA) for reliable determination of PL.
authors.affiliation: Department of Medicinal Plants Breeding and 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: Waraporn
authors.initials: W
authors.lastname: Putalun
authors.forename: Benyakan
authors.initials: B
authors.lastname: Pongkitwitoon
authors.forename: Ryota
authors.initials: R
authors.lastname: Tsuchihashi
authors.forename: Satoshi
authors.initials: S
authors.lastname: Morimoto
authors.forename: Junei
authors.initials: J
authors.lastname: Kinjo
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Shoyama
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D019684:Magnoliopsida / Q000737:chemistry*; D008969:Molecular Sequence Data; D009285:Naphthoquinones / Q000032:analysis / Q000276:immunology*; D029606:Plumbaginaceae / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D007135:Immunoglobulin Variable Region; D009285:Naphthoquinones; D011994:Recombinant Proteins; C014758:plumbagin