ANTIBODY SCFV FRAGMENTS WITHOUT DISULFIDE BONDS MADE BY MOLECULAR EVOLUTION

We generated stable and functional cysteine-free antibody single-chain fragments (scFv) lacking the conserved disulfide bonds in both V-H an d V-L. This was achieved by molecular evolution, starting from the scF v fragment of the levan binding antibody ABPC48, which is naturally mi ssing one of the conserved cysteine residues, by using DNA shuffling a nd phage display. Several of the selected sequences were expressed and the resulting scFv proteins characterized by equilibrium urea denatur ation. Three of the characterized proteins exhibit thermodynamic stabi lity similar to the wild-type protein, and these cysteine-free mutant proteins can now be expressed in functional form in the Escherichia co li cytoplasm. We believe that such molecules are of great utility for use as intrabodies, can be produced by simpler expression strategies a nd may give further insight into the folding and stability of the immu noglobulin fold. (C) 1998 Academic Press Limited.