An intrinsically stable antibody scFv fragment can tolerate the loss of both disulfide bonds and fold correctly

FEBS Lett. 1998 May 15;427(3):357-61. doi: 10.1016/s0014-5793(98)00463-3.

Abstract

A fully functional cysteine-free derivative of the intrinsically stable anti-HER2 scFv fragment hu4D5-8 was generated by replacing the disulfide forming cysteine residues in VH and VL with the amino acid combination valine-alanine in both domains. The antigen binding properties, determined by ELISA and BIAcore measurements, were not affected by removal of the disulfide bonds. The thermodynamic stability of the disulfide-containing scFv of 8.1 kcal/mol is decreased upon complete reduction of both disulfides to 2.7 kcal/mol, while that of the valine-alanine variant is somewhat higher (about 3.8 kcal/ mol). Our results suggest that, in principle, a disulfide-free fully functional derivative of any scFv can be obtained, as long as the corresponding disulfide-containing scFv has a high enough thermodynamic stability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Biosensing Techniques
  • Chromatography, Gel
  • Cysteine / chemistry
  • Disulfides / chemistry*
  • Enzyme-Linked Immunosorbent Assay
  • Immunoglobulin Fragments / biosynthesis
  • Immunoglobulin Fragments / chemistry*
  • Immunoglobulin Fragments / isolation & purification
  • Immunoglobulin Heavy Chains / biosynthesis
  • Immunoglobulin Heavy Chains / chemistry
  • Immunoglobulin Heavy Chains / isolation & purification
  • Immunoglobulin Light Chains / biosynthesis
  • Immunoglobulin Light Chains / chemistry
  • Immunoglobulin Light Chains / isolation & purification
  • Immunoglobulin Variable Region / biosynthesis
  • Immunoglobulin Variable Region / chemistry*
  • Immunoglobulin Variable Region / isolation & purification
  • Point Mutation
  • Protein Denaturation
  • Protein Folding*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Thermodynamics
  • Urea / pharmacology

Substances

  • Disulfides
  • Immunoglobulin Fragments
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Light Chains
  • Immunoglobulin Variable Region
  • Recombinant Proteins
  • immunoglobulin Fv
  • Urea
  • Cysteine