Construction and Expression of Specificity-Improved Single-Chain Variable Fragments against the Bioactive Naphthoquinone, Plumbagin

Seiichi Sakamoto; Futoshi Taura; Waraporn Putalun; Benyakan Pongkitwitoon; Ryota Tsuchihashi; Satoshi Morimoto; Junei Kinjo; Yukihiro Shoyama; Hiroyuki Tanaka

doi:10.1248/bpb.32.434

Regular Articles

Construction and Expression of Specificity-Improved Single-Chain Variable Fragments against the Bioactive Naphthoquinone, Plumbagin

Seiichi Sakamoto, Futoshi Taura, Waraporn Putalun, Benyakan Pongkitwitoon, Ryota Tsuchihashi, Satoshi Morimoto, Junei Kinjo, Yukihiro Shoyama, Hiroyuki Tanaka

Author information

Seiichi Sakamoto
Department of Medicinal Plants Breeding and Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University
Futoshi Taura
Department of Medicinal Plants Breeding and Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University
Waraporn Putalun
Faculty of Pharmaceutical Sciences, Khon Kaen University
Benyakan Pongkitwitoon
Faculty of Pharmaceutical Sciences, Khon Kaen University
Ryota Tsuchihashi
Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Fukuoka University
Satoshi Morimoto
Department of Medicinal Plants Breeding and Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University
Junei Kinjo
Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Fukuoka University
Yukihiro Shoyama
Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Nagasaki International University
Hiroyuki Tanaka
Department of Medicinal Plants Breeding and Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University

Keywords: single-chain variable fragment, plumbagin, cross-reactivity, enzyme-linked immunosorbent assay, Plumbago zeylanica

JOURNAL FREE ACCESS

2009 Volume 32 Issue 3 Pages 434-439

DOI https://doi.org/10.1248/bpb.32.434

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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 (Gly₄Ser)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.

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