Construction and expression of specificity-improved single-chain variable fragments against the bioactive naphthoquinone, plumbagin.
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.
Sakamoto
Seiichi
S
Department of Medicinal Plants Breeding and Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Taura
Futoshi
F
Putalun
Waraporn
W
Pongkitwitoon
Benyakan
B
Tsuchihashi
Ryota
R
Morimoto
Satoshi
S
Kinjo
Junei
J
Shoyama
Yukihiro
Y
Tanaka
Hiroyuki
H
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
D007135:Immunoglobulin Variable Region; D009285:Naphthoquinones; D011994:Recombinant Proteins; C014758:plumbagin