Generation of a single-chain Fv fragment for the monitoring of deoxycholic acid residues anchored on endogenous proteins.
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 (V(H)) and light (V(L)) 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 (Gly(4)Ser)(3) to construct a single scFv gene with the gene segments in the following order: 5'-V(H)-linker-V(L)-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.
Kobayashi
Norihiro
N
Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan.
Ohtoyo
Mamoru
M
Wada
Eriko
E
Kato
Yoshinori
Y
Mano
Nariyasu
N
Goto
Junichi
J
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D003840:Deoxycholic Acid / Q000737:chemistry / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006241:Haptens; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010957:Plasmids; D017433:Protein Structure, Secondary; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D006241:Haptens; D007128:Immunoglobulin Fragments; D011506:Proteins; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D003840:Deoxycholic Acid