THE DISULFIDE BONDS IN ANTIBODY VARIABLE DOMAINS - EFFECTS ON STABILITY, FOLDING INVITRO, AND FUNCTIONAL EXPRESSION IN ESCHERICHIA-COLI

The formation of the disulfide bonds in the variable domains V(H) and V(L) of the antibody McPC603 was found to be essential for the stability of all antigen binding fragments investigated. Exposure of the F(v) fragment to reducing conditions in vitro resulted in irreversible denaturation of both V(H) and V(L). In vitro refolding of the reduced F(v) fragment was only possible when the disulfide bonds were allowed to form under oxidizing conditions. The analysis of a series of mutants of the F(v) fragment, the F(ab) fragment and the single-chain F(v) fragment, all secreted into the periplasm of Escherichia coli, in which each of the cysteine residues of the variable domains was replaced by a series of other amino acids, showed that functional antigen binding fragments required the presence of both the disulfide bond in V(H) and the one in V(L). These results were also used to devise an alternative expression system based on the production of insoluble fusion proteins consisting of truncated beta-galactosidase and antibody domains, enzymatic cleavage, and refolding and assembly in vitro. This strategy should be useful for providing access to unstable antibody domains and fragments.