Generation of a single-chain Fv fragment for the monitoring of deoxycholic acid residues anchored on endogenous proteins

Norihiro Kobayashi, Mamoru Ohtoyo, Eriko Wada, Yoshinori Kato, Nariyasu Mano, Junichi Goto

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

A subset of lipophillic bile acids, including deoxycholic acid (DCA) and lithocholic acid (LCA), are thought to be biologically transformed into reactive intermediates forming covalently modified, "tissue-bound" bile acids that can exert several toxic effects. We have generated a single-chain Fv fragment (scFv) as a probe to monitor DCA residues anchored on proteins. DNA fragments encoding the variable heavy (VH) and light (VL) domains of a mouse antibody raised against a DCA hapten (Ab #88) were cloned by rapid amplification of cDNA 5′-ends. These sequences were combined via a common linker sequence coding (Gly4Ser)3 to construct a single scFv gene with the gene segments in the following order: 5′-V H-linker-VL-3′. This construct was subcloned into an antibody-expression vector, pEXmide 5; soluble scFv protein was then expressed in the bacterial periplasm of the XLOLR Escherichia coli strain. In a competitive enzyme-linked immunosorbent assay using DCA-coated microtiter plates, the scFv provided a dose-response curve for free DCA ranging between 2 and 5000 pg/assay. The scFv reacts similarly with the l-lysine adduct of DCA (cross-reactivity, 72%), while bile acids having a modified DCA steroid skeleton were well-discriminated (cross-reactivity, <1%). This scFv could also monitor trace amounts of DCA residues anchored on a protein through DCA acyl adenylate reactions, the likely reactive intermediate. The present scFv may be a useful tool for trace characterization of tissue-bound bile acids; this usefulness may be significantly enhanced by fusion with signal-generating proteins, such as alkaline phosphatase or green fluorescent protein.

Original languageEnglish
Pages (from-to)285-294
Number of pages10
JournalSteroids
Volume70
Issue number4
DOIs
Publication statusPublished - 2005 Apr

Keywords

  • 5′RACE
  • Bile acid
  • Deoxycholic acid
  • Single-chain Fv fragment
  • Tissue-bound bile acid
  • V-gene cloning

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Endocrinology
  • Pharmacology
  • Clinical Biochemistry
  • Organic Chemistry

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