The disulfide bonds in antibody variable domains: effects on stability, folding in vitro, and functional expression in Escherichia coli.
The formation of the disulfide bonds in the variable domains VH and VL of the antibody McPC603 was found to be essential for the stability of all antigen binding fragments investigated. Exposure of the Fv fragment to reducing conditions in vitro resulted in irreversible denaturation of both VH and VL. In vitro refolding of the reduced Fv fragment was only possible when the disulfide bonds were allowed to form under oxidizing conditions. The analysis of a series of mutants of the Fv fragment, the Fab fragment and the single-chain Fv 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 VH and the one in VL. 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.
Glockshuber
R
R
Genzentrum, Universität München, Max-Planck-Institut für Biochemie, Martinsried, Germany.
Schmidt
T
T
Plückthun
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001666:Binding Sites, Antibody; D002417:Cattle; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D010957:Plasmids; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D013329:Structure-Activity Relationship
D004220:Disulfides; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D003545:Cysteine