Activation of a Refolded, Berberine-specific, Single-chain Fv Fragment by Addition of Free Berberine |
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| Authors: | Jun-Sik Kim Tadashi Masaki Supaart Sirikantaramas Yukihiro Shoyama Hiroyuki Tanaka |
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| Affiliation: | (1) Department of Oriental Medicine Resources, College of Health and Welfare, Dong-Shin University, 252 Daeho-dong, Naju, Jeonnam, 520-714, Republic of Korea;(2) Department of Medicinal Resources Regulation, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan |
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| Abstract: | 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 (VH) and light chain (VL) genes were connected with a flexible linker using an assembly PCR. The VH-linker-VL 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. |
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| Keywords: | Artificial chaperone Berberine ELISA Inclusion body Refolding scFvs |
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