Activation of a refolded, berberine-specific, single-chain Fv fragment by addition of free berberine

Biotechnol Lett. 2006 Jul;28(13):999-1006. doi: 10.1007/s10529-006-9033-7. Epub 2006 Jun 20.

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 (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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal / immunology*
  • Antibodies, Monoclonal / metabolism*
  • Berberine / immunology*
  • Cell Line
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Humans
  • Hybridomas / metabolism
  • Immunoglobulin Variable Region / genetics
  • Immunoglobulin Variable Region / immunology*
  • Mice
  • Mice, Inbred BALB C
  • Protein Engineering / methods*
  • Protein Folding

Substances

  • Antibodies, Monoclonal
  • Immunoglobulin Variable Region
  • Berberine