Relative position of the hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct

Biochim Biophys Acta. 2000 Sep 1;1523(1):13-20. doi: 10.1016/s0304-4165(00)00086-6.


Hexahistidine tag (His-tag) is the most widely used tag for affinity purification of recombinant proteins for their structural and functional analysis. In the present study, single chain Fv (scFv) constructs were engineered form the monoclonal antibody (MAb) CC49 which is among the most extensively studied MAb for cancer therapy. For achieving efficient purification of scFvs by immobilized metal-ion affinity chromatography (IMAC), a His-tag was placed either at the C-terminal (scFv-His6) or N-terminal (His6-scFv) of the coding sequence. Solid-phase radioimmunoassay for scFv-His6 showed only 20-25% binding whereas both His6-scFv and scFv (no His-tag) showed 60-65% binding. Surface plasmon resonance studies by BIAcore revealed the binding affinity constant (KA) for His6-scFv and scFv as 1.19 x 10(6) M(-1) and 3.27 x 10(6) M(-1), respectively. No K(A) value could be calculated for scFv-His6 due to poor binding kinetics (kon and koff). Comparative homology modeling for scFv and scFv-His6 showed that the C-terminal position of the His-tag partially covered the antigen-binding site of the protein. The study demonstrates that the C-terminal position of His-tag on the CC49 scFv adversely affects the binding properties of the construct. The results emphasize the importance of functional characterization of recombinant proteins expressed with purification tags.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / chemistry
  • Antibodies, Monoclonal / immunology
  • Binding Sites, Antibody*
  • Cattle
  • Chromatography, High Pressure Liquid
  • Cloning, Molecular
  • Enzyme-Linked Immunosorbent Assay
  • Escherichia coli
  • Histidine
  • Immunoglobulin Fragments / chemistry*
  • Immunoglobulin Fragments / immunology
  • Models, Molecular
  • Mucins / chemistry*
  • Mucins / immunology*
  • Oligopeptides
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / immunology


  • Antibodies, Monoclonal
  • Immunoglobulin Fragments
  • Mucins
  • Oligopeptides
  • Recombinant Proteins
  • immunoglobulin Fv
  • Histidine