Activation of a refolded, berberine-specific, single-chain Fv fragment by addition of free berberine.
A single-chain variable fragment (scFv) specific for berberine was produced in Escherichia coli. The anti-berberine scFv gene was cloned from hybridoma 1D5-3B-7 producing the monoclonal antibody. The variable regions of the heavy (V(H)) and light chain (V(L)) genes were connected with a flexible linker using an assembly PCR. The V(H)-linker-V(L) gene was inserted into a plasmid, pET28a (+), then overexpressed in E. coli BL21 (DE3). The active of the scFv by refolding based on stepwise dialysis methods and an artificial chaperone was determined by direct and competitive enzyme-linked immunosorbent assay (ELISA). The results of direct ELISA showed that the anti-berberine scFv retained specific binding activity to berberine. In competitive ELISA, however, activity was increased depending on the concentration of berberine.
Kim
Jun-Sik
JS
Department of Oriental Medicine Resources, College of Health and Welfare, Dong-Shin University, 252 Daeho-dong, Naju, Jeonnam 520-714, Republic of Korea.
Masaki
Tadashi
T
Sirikantaramas
Supaart
S
Shoyama
Yukihiro
Y
Tanaka
Hiroyuki
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D001599:Berberine / Q000276:immunology*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D001599:Berberine