Disulfide bonds contribute greatly to the stability of antibody immunoglobulin domains. Under reducing conditions, such as those that prevail in the cytoplasm, disulfide bonds do not normally form and as a result most antibodies expressed in that compartment accumulate in a misfolded and inactive state. We have developed a simple method for the quantitative isolation of antibody fragments that retain full activity under reducing conditions from large mutant libraries. In E. coli, inactivation of the cysteine oxidoreductase DsbA abolishes protein oxidation in the periplasm leads to the accumulation of scFvs and other disulfide-containing proteins in a reduced form. Libraries of mutant scFvs were tethered onto the inner membrane in periplasm of dsbA(-) cells and mutants that could bind fluorescently labeled antigen in the reducing periplasm were screened by Anchored Periplasmic Expression. Using this approach, scFv antibody variants could be isolated which are fully active when expressed in the cytoplasm or when the four Cys residues that are normally required for disulfide bond formation are substituted by Ser residues

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