Article
Received: 5 September 2008; Revised: 29 October 2008; Accepted: 2 November 2008
10.1002/pro.31 About DOI
Engineering antibody fragments to fold in the absence of disulfide bonds |
| Min Jeong Seo 1 2, Ki Jun Jeong 6, Clinton E. Leysath 2, Andrew D. Ellington 2 3, Brent L. Iverson 2 3, George Georgiou 1 2 4 5 * |
| 1Department of Chemical Engineering, University of Texas, Austin, TX 78712 2Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712 3Department of Chemistry and Biochemistry, University of Texas, Austin, TX 78712 4Section of Molecular Genetics and Microbiology, University of Texas, Austin, TX 78712 5Department of Biomedical Engineering, University of Texas, Austin, TX 78712 6Department of Chemical and Biomolecular Engineering Institute for the Bio Century, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea |
| email: George Georgiou (gg@che.utexas.edu) |
*Correspondence to George Georgiou, Department of Chemical Engineering, Cockrell School of Engineering, The University of Texas at Austin, 1 University Station C0400, Austin, TX 78712
Funded by:
ONR DARPA
| Keywords |
| protein structure/folding disulfide bonds directed evolution intrabodies |
| Abstract |
Disulfide bonds play a critical role in the stabilization of the immunoglobulin  -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. |
Received: 5 September 2008; Revised: 29 October 2008; Accepted: 2 November 2008
| Digital Object Identifier (DOI) |
10.1002/pro.31 About DOI
