Engineering antibody fragments to fold in the absence of disulfide bonds

Abstract

Disulfide bonds play a critical role in the stabilization of the immunoglobulin b-sandwich sandwich. 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 (intrabodies) 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, which 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 of dsbA cells and mutants that could bind fluorescently labeled antigen in the reducing periplasm were screened by Anchored Periplasmic Expression (APEx; Harvey et al., Proc Natl Acad Sci USA 2004;101:9193-9198.). Using this approach, we isolated scFv antibody variants that are fully active when expressed in the cytoplasm or when the four Cys residues that normally form disulfides are substituted by Ser residues.

Immunoglobulin domains inEscherichia coliand other enterobacteria: from pathogenesis to applications in antibody technologies

“…Functional Ab fragments have also been expressed in the cytoplasm of E. coli cells. Interestingly, APEx has been employed for selection of stable scFvs able to fold in reducing environments taking advantage of their expression in the periplasm of E. coli dsbA mutants (Seo et al, 2009), in which oxidation of disulfide bonds in periplasmic Ab fragments is abolished (Jurado et al, 2002). Lack of disulfide bonds in the cytoplasm of E. coli is owed to the thioredoxin and glutaredoxin pathways that keep free thiol groups in a reduced state (Ritz & Beckwith, 2001;Kadokura et al, 2003).…”

“…Intrabodies selected in vivo with E. coli trxB gor cells may be stabilized later for correct folding in the reducing environment of the cytoplasm of wild-type cells. Interestingly, APEx has been employed for selection of stable scFvs able to fold in reducing environments taking advantage of their expression in the periplasm of E. coli dsbA mutants (Seo et al, 2009), in which oxidation of disulfide bonds in periplasmic Ab fragments is abolished (Jurado et al, 2002). Using this methodology highly stable scFvs that fold in the periplasm of E. coli dsbA mutants were selected and shown to be active when expressed in the cytoplasm of wild-type E. coli cells.…”

GFP‐SCFV: Expression and possible applications as a tool for experimental investigations of atherosclerosis

“…To stabilize its folding, GFP‐scFv requires the formation of two disulfide bonds, one in the heavy chain and one in the light chain of scFv. It is well known that disulfide bonds are formed by the oxidation reaction between two cysteine residues; however, as the cytoplasm of the bacterium is a reducing environment, disulfide bridges are not formed, and the protein tends to accumulate as inclusion bodies . Nevertheless, inclusion bodies are not necessarily disadvantageous to the overall process.…”

“…It is well known that disulfide bonds are formed by the oxidation reaction between two cysteine residues; however, as the cytoplasm of the bacterium is a reducing environment, disulfide bridges are not formed, and the protein tends to accumulate as inclusion bodies. 39 Nevertheless, inclusion bodies are not necessarily disadvantageous to the overall process. Inclusion bodies tend to be less toxic to the host cell and are protected from proteolytic digestion.…”

Recombinant antibodies: Engineering and production in yeast and bacterial hosts

“…In this work, in addition to Trx fusion, co-expression of cytoplasmic molecular chaperons (GroELS and trigger factors) increased the specific productivity of an antibody fragment almost threefold.The reducing cytoplasm itself is the preferable space for the production of intrabodies, which do not require disulfide bond formation for their activity (route 4). Using HTS based on new protein display (APEx 2-hybrid) systems in which target antibodies are displayed on periplasmic side of inner membrane via lipoprotein fusion in E. coli, new intrabodies were isolated, and a three-to fivefold increase in production yields was exhibited compared to that of the original antibody fragment [49]. So far, despite the distinctive advantage associated with simple recovery of antibodies from the culture medium, only few examples of antibody production into the extracel-lular compartment have been described (route 10).…”