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

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 V-H and V-L 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 kcallmol, 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. (C) 1998 Federation of European Biochemical Societies.