Engineering antibody fragments to fold in the absence of disulfide bonds |
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Authors: | Min Jeong Seo Ki Jun Jeong Clinton E Leysath Andrew D Ellington Brent L Iverson George Georgiou |
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Institution: | 1. Department of Chemical Engineering, University of Texas, Austin, TX 78712;2. Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712;3. Department of Chemical and Biomolecular Engineering Institute for the Bio Century, KAIST, 373‐1 Guseong‐dong, Yuseong‐gu, Daejeon 305‐701, Korea;4. Department of Chemistry and Biochemistry, University of Texas, Austin, TX 78712;5. Section of Molecular Genetics and Microbiology, University of Texas, Austin, TX 78712;6. Department of Biomedical Engineering, University of Texas, Austin, TX 78712 |
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Abstract: | Disulfide bonds play a critical role in the stabilization of the immunoglobulin β-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. |
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Keywords: | protein structure/folding disulfide bonds directed evolution intrabodies |
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